Increasing fermentation kinetics in a recombinant yeast

Abstract

The present disclosure provides a recombinant yeast host cell for converting a carbohydrate into a fermentation product with an increased fermentation kinetic. The recombinant yeast host cell comprises an upregulated pyruvate kinase, a first engineered metabolic pathway to convert the carbohydrate into acetyl-coA comprising a heterologous polypeptide having phosphoketolase activity, and a second engineered metabolic pathway to convert acetyl-CoA into the fermentation product. The fermentation kinetic is increased when compared to a recombinant yeast host comprising the first and second engineered pathway but lacking the upregulated pyruvate kinase.

Description

[0001] INCREASING FERMENTATION KINETICS IN A RECOMBINANT YEAST

[0002] CROSS-REFERENCE TO RELATED APPLICATION(S) AND DOCUMENT(S)

[0003] This patent application claims priority to US provisional application 63 / 700,998 filed on September 30, 2024 and herewith incorporated in its entirety. This application also comprises a sequence listing in electronic form which is also incorporated in its entirety.

[0004] TECHNOLOGICAL FIELD

[0005] The present disclosure concerns a recombinant yeast host cell comprising an engineered pathways to convert carbohydrate into a fermentation product comprising phosphoketolase activity and process thereof.

[0006] BACKGROUND

[0007] Saccharomyces cerevisiae is utilized as the primary biocatalyst in commercial bioethanol production. S. cerevisiae can be engineered to generate higher value chemical products within corn ethanol fermentations either in place of ethanol or co-produced with ethanol. Although good progress has been made on increasing fermentation product titers, the fermentation kinetics of the engineered strains need, in some instance, to be improved for the strain to perform well in a commercial setting.

[0008] There is thus a need in providing alternatives to increase fermentation kinetics of strains engineered with a pathway to convert carbohydrates into fermentation products.

[0009] BRIEF SUMMARY

[0010] The present disclosure concerns a recombinant yeast host cell having an engineered pathways to convert carbohydrate to a fermentation product and having an increased fermentation kinetic.

[0011] According to a first aspect, the present disclosure provides a recombinant yeast host cell for converting a carbohydrate into a fermentation product comprising a) an upregulated pyruvate kinase; b) a first engineered metabolic pathway to convert the carbohydrate into acetyl-coA comprising a heterologous polypeptide having phosphoketolase activity; and c) a second engineered metabolic pathway to convert acetyl-CoA into the fermentation product. In one embodiment, the upregulated pyruvate kinase is a heterologous pyruvate kinase. In more specific embodiment, the heterologous pyruvate kinase is derived from a Saccharomyces cerevisiae pyruvate kinase. In some embodiment, the heterologous pyruvate kinase is a pyruvate kinase 1 (PYK1) or a pyruvate kinase 2 (PYK2). In another embodiment, the heterologous pyruvate kinase has the amino acid sequence of SEQ ID NO: 312, 314 or 316, or is a variant of the SEQ ID NO: 312, 314 or 316 having a pyruvate kinase activity. In one embodiment, the heterologous polypeptide having phosphoketolase activity is a phosphoketolase classified under Enzyme Commission No. 4.1.2.9 or 4.1.2.22; has the ability to convert D-xylulose 5-phosphate into D-glyceraldehyde 3- phosphate and acetyl-phosphate; has the ability to convert D-fructose 6-phosphate into D-erythrose 4-phosphate; has the ability to convert D-sedoheptulose 7-phosphate into D- ribose 5-phosphate; has single- or multiple-specificity; is of prokaryotic or eukaryotic origin; is encoded by a phk1 gene or a phk2 gene; is derived from Bifidobacterium, Lactiplantibacillus, Leuconostoc, Penicillium, Aspergillus, Oenococcus, Clostridium or Neurospora species; is derived from Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium gallicum, Bifidobacterium animalis, Bifidobacterium adolescentis, Bifidobacterium asteroides, Bifidobacterium longum, Lactobacillus pentosus, Lactobacillus acidophilus, Lactobacillus casei, Lactiplantibacillus plantarum, Penicillium chrysogenum, Aspergillus niger, Aspergillus nidulans, Aspergillus clavatus, Neurospora crassa, Leuconostoc mesenteroides, Clostridium acetobutylicum or Oenococcus oenr, has the amino acid sequence of SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 or 70, or is a variant of the SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, or 344 having a phosphoketolase activity; and / or is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, or 343 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, or 344. In some other embodiment, the heterologous phosphoketolase has at least the ability to convert D-fructose 6-phosphate into D-erythrose 4-phosphate. In one embodiment, the fermentation product comprises acetone, isopropanol, and / or ethanol. In some embodiment, the fermentation product comprises acetone and / or isopropanol. In one embodiment, the second engineered pathway comprises a thiolase; a CoA transferase, a HMG-CoA synthase and lyase, or an acetoacetyl-CoA hydrolase; an acetoacetate decarboxylase; and optionally a first alcohol dehydrogenase. In another embodiment, the thiolase is heterologous; of prokaryotic or eukaryotic origin; encoded by a th! gene, an erg10 gene or a phaA gene; derived from Clostridium, Saccharomyces, Cupriavidus, Clostridium, Yarrowia, Thermoanaerobacterium, Saccoglossus, Strongylocentrotus, Zygosaccharomyces or Paenibacillus species; derived from Clostridium acetobutylicum, Clostridium beijerinckii, Saccharomyces cerevisiae, Cupriavidus necator, Clostridium kluyveri, Yarrowia lipolytica, Thermoanaerobacterium thermosaccharolyticum, Saccogiossus kowalevskii, Strongylocentrotus purpuratus, Zygosaccharomyces bailii or Paenibacillus polymyxa has the amino acid sequence of SEQ ID NO: 144, 162, 164, 166, 168, 170, 172, 174, 176, 178 or 180, or is a variant of the SEQ ID NO: 144, 162, 164, 166, 168, 170, 172, 174, 176, 178 or 180 having a thiolase activity; and / or is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 143, 161 , 163, 165, 167, 169, 171 , 173, 175, 177 or 179, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 144, 162, 164, 166, 168, 170, 172, 174, 176, 178 or 180. In another embodiment, the CoA transferase is heterologous; of prokaryotic or eukaryotic origin; encoded by a ctfA gene, a ctfB gene, an atoD and / or an atoA gene; derived from Clostridium, Thermosipho, Escherichia, Paenibacillus, Alkaliphilus or Brevibacillus species; derived from Clostridium acetobutylicum, Thermosipho melanesiensis, Escherichia coli, Paenibacillus polymyxa, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum, Clostridium sticklandii, Alkaliphilus metalliredigens, or Clostridium bovifaecis or Brevibacillus laterosporus has the amino acid sequence of SEQ ID NO:146, 148, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214 or 216, or is a variant of the SEQ ID NO: 146, 148, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214 or 216, having a CoA transferase activity; and / or is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 145, 147, 185, 187, 189, 191 , 193, 195, 197, 199, 201 , 203, 205, 207, 209, 211 , 213 or 215, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 146, 148, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214 or 216. In one embodiment, the HMG-CoA synthase HMG-CoA synthase is: heterologous; of prokaryotic or eukaryotic origin; encoded by a hgms gene or an erg13 gene; derived from Saccharomyces, Lacticaseibacillus, Enterococcus, Haloferax, Alloscardovia or Listeria’, derived from Saccharomyces cerevisiae, Lacticaseibacillus casei, Enterococcus faecalis, Haloferax volcanii, Alloscardovia theropitheci, or Listeria monocytogenes-, has the amino acid sequence of SEQ ID NO: 240, 242, 244, 246, 248 or 250, or is a variant of the SEQ ID NO: 240, 242, 244, 246, 248 or 250 having a HMG-CoA synthase activity; and / or is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 239, 241 , 243, 245, 247 or 249, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ I D NO: 240, 242, 244, 246, 248 or 250. In another embodiment, the HMG-CoA lyase is heterologous; of prokaryotic or eukaryotic origin; encoded by an hmgcl gene; derived from Pseudomonas, Azotobacter, Bacillus, Desulfotomaculum, Acinetobacter, Moraxella, Alcaligenaceae, Macaca, Arabidopsis, Gallus, or Danio species’, derived from Pseudomonas monteilii, Pseudomonas wayambapalatensis, Azotobacter vinelandii, Pseudomonas citronellolis, Pseudomonas cremoris, Pseudomonas chengduensis, Pseudomonas aeruginosa, Bacillus subtilis, Desulfotomaculum arcticum, Desulfoscipio geothermicus, Acinetobacter baumannii, Acinetobacter Iwoffii, Moraxella caviae, Alcaligenaceae bacterium, Macaca fascicularis, Arabidopsis thaliana, Gallus gallus, or Danio ren , has the amino acid sequence of SEQ ID NO: 252, 254, 256, 258, 260, 262, 263, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, or 286, or is a variant of the SEQ ID NO: 252, 254, 256, 258, 260, 262, 263, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, or 286having a HMG-CoA lyase activity; and / or is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 251 , 253, 255, 257, 259, 261 , 263, 265, 267, 269, 271 , 273, 275, 277, 279, 281 , 283 or 285, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 252, 254, 256, 258, 260, 262, 263, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, or 286. In one embodiment, the acetoacetyl-CoA hydrolase is: heterologous; of prokaryotic or eukaryotic origin; encoded by a tesB gene, yciA gene, a yigl gene, a ydil gene, a fadM1 gene, a fadM2 gene or a fadM gene; derived from Escherichia, Methylorubrum, Pseudomonas, Campylobacter, Mycobacterium, Fibrobacter, Alcanivorax, Haemophilus, Zymomonas, Providencia or Prevotella derived from Escherichia coll, Methylorubrum extorquens, Pseudomonas aeruginosa, Campylobacter jejuni, Mycobacterium tuberculosis, Pseudomonas putida, Fibrobacter succinogenes, Alcanivorax borkumensis, Haemophilus influenzae, Zymomonas mobilis subsp. Mobilis ZM4, Campylobacter jejuni, Prevotella ruminicola or Providencia sneebia and / or has the amino acid sequence of SEQ ID NO: 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302 or 303, or is a variant of the SEQ ID NO: 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302 or 303 having an acetoacetyl-CoA hydrolase activity; and / or is encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302 or 303. In another embodiment, the acetoacetate decarboxylase is heterologous; of prokaryotic or eukaryotic origin; encoded by an adc gene;derived from Clostridium, Bacillus, Lacticaseibacillus, Rhizobium, Bradyrhizobium, Tetrahymena, Aspergillus or Paenibacillus species; derived from Clostridium acetobutylicum, Clostridium beijerinckii, Bacillus amyloliquefaciens, Lacticaseibacillus casei, Lacticaseibacillus rhamnosus, Rhizobium leguminosarum, Bradyrhizobium japonicum, Tetrahymena thermophile, Aspergillus bertholletiae, Aspergillus niger or Paenibacillus polymyxa has the amino acid sequence of SEQ ID NO: 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, or 238, or is a variant of the SEQ ID NO: 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, or 238 having an acetoacetate decarboxylase activity; and / or is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 217, 219, 221 , 223, 225, 227, 229, 231 , 233, 235 or 237, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, or 238. In one embodiment, the first alcohol dehydrogenase is: native and / or heterologous; of prokaryotic or eukaryotic origin; encoded by an adh gene; derived from Zymomonas or Saccharomyces species; derived from Saccharomyces cerevisiae or Zymomonas mobilise has the amino acid sequence of SEQ ID NO: 304, 305, or 306 or is a variant of the SEQ ID NO: 304, 305, or 306 having an alcohol dehydrogenase activity; and / or is encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 304, 305, or 306. In another embodiment, the first engineered metabolic pathway further comprises a heterologous phosphotransacetylase, a heterologous acetate kinase, and / or a heterologous acetyl CoA synthetase. In some embodiment, the heterologous phosphotransacetylase is:of prokaryotic or eukaryotic origin encoded by a pta gene; derived from Bifidobacterium, Leuconostoc, Oenococcus, Azotobacter, Lacticaseibacillus, Bacillus, Salmonella, Clostridium, Phytophthora, Globisporangium or Holophagae species; derived from Bifidobacterium adolescentis, Bifidobacterium animalis, Leuconostoc mesenteroides, Oenococcus oenii, Azotobacter vinelandii, Lactiplantibacillus plantarum, Bacillus subtilis, Salmonella enterica, Clostridium kluyveri, Clostridium phytofermentans, Phytophthora ramorum, Globisporangium splendens or Holophagae bacterium’, has the amino acid sequence of SEQ ID NO: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 or 102, or is a variant of the SEQ ID NO: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 or 102 having a phosphotransacetylase activity; and / or is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 75, 77, 79, 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99, or 101 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 or 102. In some embodiment, the heterologous acetate kinase is: of prokaryotic or eukaryotic origin; encoded by an ack gene; derived from Bifidobacterium, Leuconostoc, Oenococcus, Escherichia, Bacillus, Clostridium, Salmonella, Phytophthora, Chlamydomonas, Aspergillus or Lactiplantibacillus, species; derived from Bifidobacterium adolescentis, Leuconostoc mesenteroides, Oenococcus oenii, Escherichia coli, Bacillus subtilis, Clostridium acetobutylicum, Salmonella enterica, Phytophthora ramorum, Chlamydomonas reinhardtii, Aspergillus nidulans, Lactiplantibacillus plantarum or Clostridium kluveryr, has the amino acid sequence of SEQ ID NO: 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128 or 130, or is a variant of the SEQ ID NO: 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128 or 130 having an acetate kinase activity; and / or is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 107, 109, 111 , 113, 115, 117, 119, 121 , 123, 125, 127 or 129, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128 or 130. In some embodiment, the heterologous acetyl CoA synthetase is of prokaryotic or eukaryotic origin; encoded by an acs1 gene or an acs2 gene; derived from Saccharomyces, Zygosaccharomyces, Salmonella, Acetobacter or Escherichia species’, derived from Saccharomyces cerevisiae, Zygosaccharomyces bailii, Salmonella enterica, Acetobacter aceti or Escherichia coir, and / or has the amino acid sequence of SEQ ID NO: 132, 134, 136, 138, 140 or 142, or is a variant of the SEQ ID NO: 132, 134, 136, 138, 140 or 142 having an acetyl CoA synthetase activity; and / or is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 131 , 133, 135, 137, 139 or 141 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 132, 134, 136, 138, 140 or 142. In one embodiment, the recombinant yeast host cell being from the genus Saccharomyces sp. In some embodiment, the recombinant yeast host cell being from the species Saccharomyces cerevisiae. In one embodiment, the recombinant yeast host cell is having an increased fermentation kinetic when compared to a control yeast host cell comprising the first and the second engineered metabolic pathways as defined in b) and c) but lacking the upregulated pyruvate kinase as defined in a). In some other embodiment, the recombinant yeast host cell is producing higher fermentation product yield than a control yeast host cell comprising the first and the second engineered metabolic pathways as defined in b) and c) but lacking the upregulated pyruvate kinase as defined in a).

[0012] In a second aspect, the present disclosure provides a process for making a fermentation product from carbohydrate. The process comprises contacting the recombinant yeast host cell with a carbohydrate source under a condition allowing the conversion of at least a part of the carbohydrate into a fermentation product. In one embodiment, the carbohydrate source comprises a corn mash. In another embodiment, the recombinant yeast host cell has an increased fermentation kinetic when compared to a control yeast host cell comprising the first and the second engineered metabolic pathway as defined in b) and c) but lacking the upregulated pyruvate kinase as defined in a).

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Having thus generally described the nature of the invention, reference will now be made to the accompanying drawings, showing by way of illustration, a preferred embodiment thereof, and in which:

[0015] Figure 1 provides an embodiment of a metabolic pathway using the first and second engineered pathways of the present disclosure for converting carbohydrates into acetone.

[0016] Figure 2 provides CO2production profiles from corn mash fermentation of non genetically modified S. cerevisiae strain (SC-1), an acetone producer engineered S. cerevisiae strain (SC-2), and acetone producer engineered S. cerevisiae strains further modified by upregulating pyruvate kinase activity with overexpression of wild-type PYK1 gene (SC-3) or PYK1E392Avariant (SC-4).

[0017] Figure 3 provides the metabolites and residual glucose profiles following fermentation by strains SC-1, SC-2, SC-3, and SC-4 of corn mash medium. All values are shown as g / L of glucose, glycerol, acetone, and ethanol in function of the strain / isolate tested.

[0018] Figure 4 provides CO2production rate profiles from corn mash fermentation of non- genetically modified S. cerevisiae strain (SC-1), an acetone producer engineered S. cerevisiae strains further modified by upregulating pyruvate kinase activity with overexpression of wild-type PYK1 (SC-5) or with PYK1 upregulation removed (SC-6).

[0019] Figure 5 provides the metabolites and residual glucose profiles following fermentation by strains SC-1, SC-5 and SC-6 of corn mash medium. All values are shown as g / L of glucose, glycerol, acetone, and ethanol in function of the strain / isolate tested.

[0020] DETAILED DESCRIPTION

[0021] The present disclosure concerns a recombinant yeast host cell for converting carbohydrates into a fermentation product having an increased fermentation kinetic. Amongst other things, the recombinant yeast host cell comprises at least (a) an upregulated pyruvate kinase, (b) a first engineered metabolic pathway to convert the carbohydrate into acetyl-coA comprising a heterologous polypeptide having phosphoketolase activity, and (c) a second engineered metabolic pathway to convert acetyl-CoA into the fermentation product. The fermentation kinetic observed when culturing the recombinant yeast host cell is increased when compared to a control yeast host cell comprising the first and the second engineered metabolic pathways as defined in (b) and (c) but lacking the upregulated pyruvate kinase as defined in (a).

[0022] Recombinant yeast host cells for making a fermentation product

[0023] The present disclosure provides a recombinant yeast host cell. These recombinant yeast host cells can be obtained by introducing one or more genetic modifications in a corresponding native (parental) yeast host cell.

[0024] The one or more additional genetic modifications can be made in the coding portion or the non-coding portion of a gene. For example, the recombinant yeast host cells can be obtained by introducing one or more genetic modifications in a corresponding native (parental) yeast host cell. When the genetic modification is aimed at reducing or inhibiting the expression of a specific targeted gene (which is endogenous / native to the host cell), the genetic modifications can be made in one or all copies of the targeted gene(s). When the genetic modification is aimed at increasing the expression of a specific targeted gene, the genetic modification can be made in one or multiple genetic locations. When the genetic modification is aimed at increasing the expression of a specific targeted gene, the genetic modification can be made in one or more regulatory sequence of a native to increase its expression. When the genetic modification is aimed at increasing the expression of a specific targeted gene, the genetic modification can be the addition of one or more copies of the heterologous gene encoding a heterologous polypeptide in the genome of the recombinant host. In the context of the present disclosure, when a yeast host cell is qualified as being “genetically engineered”, it is understood to mean that it has been manipulated to either add at least one or more heterologous or exogenous nucleic acid residue and / or removed at least one endogenous (or native) nucleic acid residue. In some embodiments, the one or more nucleic acid residues that are added can be derived from a heterologous cell or the recombinant host cell itself. In the latter scenario, the nucleic acid residue(s) is (are) added at a genomic location which is different than the native genomic location. The genetic manipulations did not occur in nature and are the results of in vitro manipulations of the native yeast host cell. When expressed in recombinant yeast host cells, the heterologous polypeptides described herein are encoded on one or more heterologous nucleic acid molecule. The term “heterologous” when used in reference to a nucleic acid molecule (such as a promoter or a coding sequence) refers to a nucleic acid molecule that is not natively found in the recombinant host cell. “Heterologous” also includes a native coding region, or portion thereof, that is removed from the source organism and subsequently reintroduced into the source organism in a form that is different from the corresponding native gene, e.g., not in its natural location in the organism's genome or as additional copies at its natural location. The heterologous nucleic acid molecule is purposively introduced into the recombinant yeast host cell. In some embodiments, the term “heterologous” as used herein also refers to an element (nucleic acid or protein) that is derived from a source other than the endogenous source. Thus, for example, a heterologous element could be derived from a different strain of host cell, or from an organism of a different taxonomic group (e.g., different kingdom, phylum, class, order, family genus, or species, or any subgroup within one of these classifications).

[0025] The heterologous nucleic acid molecule present in the recombinant yeast host cell can be integrated in the host cell’s genome. The term “integrated” as used herein refers to genetic elements that are placed, through molecular biology techniques, into the chromosome of a yeast host cell. For example, genetic elements can be placed into the chromosome(s) of the host cell as opposed to in a vector such as a plasmid carried by the host cell. Methods for integrating genetic elements into the genome of a host cell are well known in the art and include homologous recombination. The heterologous nucleic acid molecule can be present in one or more copies in the yeast host cell’s chromosome. The heterologous nucleic acid molecule can be integrated at a neutral integration site. Alternatively, the heterologous nucleic acid molecule can be independently replicating from the yeast host cell’s chromosome. In such embodiment, the nucleic acid molecule can be stable and self-replicating.

[0026] In some embodiments, heterologous nucleic acid molecules which can be introduced into the recombinant yeast host cells are codon-optimized with respect to the intended recipient recombinant yeast host cell. As used herein the term “codon-optimized coding region” means a nucleic acid coding region that has been adapted for expression in the cells of a given organism by replacing at least one, or more than one, codons with one or more codons that are more frequently used in the genes of that organism. In general, highly expressed genes in an organism are biased towards codons that are recognized by the most abundant tRNA species in that organism. One measure of this bias is the “codon adaptation index” or “CAI,” which measures the extent to which the codons used to encode each amino acid in a particular gene are those which occur most frequently in a reference set of highly expressed genes from an organism. The CAI of codon optimized heterologous nucleic acid molecule described herein corresponds to between about 0.8 and 1.0, between about 0.8 and 0.9, or about 1.0. In some embodiments, heterologous nucleic acid molecules which can be introduced into the recombinant host cells are codon- optimized with respect to the intended recipient recombinant host cell so as to limit or prevent homologous recombination with the corresponding native gene.

[0027] The heterologous nucleic acid molecules of the present disclosure can comprise a coding region for one or more polypeptides to be expressed by the recombinant yeast host cell. The DNA or RNA “coding region” of a heterologous nucleic acid molecule is a DNA or RNA molecule which is transcribed and / or translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. “Suitable regulatory regions” refer to nucleic acid regions located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region, and which influence the transcription, RNA processing or stability, or translation of the associated coding region. Regulatory regions may include promoters, translation leader sequences, RNA processing sites, effector binding sites and stem-loop structures. The boundaries of the coding region are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding region can include, but is not limited to, prokaryotic regions, cDNA from mRNA, genomic DNA molecules, synthetic DNA molecules, or RNA molecules. If the coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding region. In an embodiment, the coding region can be referred to as an open reading frame. “Open reading frame" is abbreviated ORF and means a length of nucleic acid, either DNA, cDNA or RNA, that comprises a translation start signal or initiation codon, such as an ATG or AUG, and a termination codon and can be potentially translated into a polypeptide sequence.

[0028] The nucleic acid molecule(s) described herein can comprise a non-coding region, for example a transcriptional and / or translational control regions. “Transcriptional and translational control regions” are DNA regulatory regions, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding region in a host cell. In eukaryotic cells, polyadenylation signals are control regions.

[0029] The heterologous nucleic acid molecule(s) can be introduced in the host cell using a vector. A “vector,” e.g., a “plasmid”, “cosmid” or “artificial chromosome” (such as, for example, a yeast artificial chromosome) refers to an extra chromosomal element and is usually in the form of a circular double-stranded DNA molecule. Such vectors may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a host cell.

[0030] In the heterologous nucleic acid molecules described herein, the promoter and the nucleic acid molecule coding for the one or more polypeptides (such as the one or more enzymes) can be operatively linked to one another. In the context of the present disclosure, the expressions “operatively linked” or “operatively associated” refers to fact that the promoter is physically associated to the nucleotide acid molecule coding for the one or more polypeptides in a manner that allows, under certain conditions, for expression of the one or more polypeptides from the heterologous nucleic acid molecule. In an embodiment, the promoter can be located upstream (5’) of the nucleic acid sequence coding for the one or more polypeptide. In still another embodiment, the promoter can be located downstream (3’) of the nucleic acid sequence coding for the one or more polypeptide. In the context of the present disclosure, one or more than one promoter can be included in the heterologous nucleic acid molecule. When more than one promoters are included in the heterologous nucleic acid molecule, each of the promoters is operatively linked to the nucleic acid sequence coding for the one or more polypeptide. The promoters can be located, in view of the nucleic acid molecule coding for the one or more protein, upstream, downstream as well as both upstream and downstream.

[0031] “Promoter” refers to a DNA fragment capable of controlling the expression of a coding sequence or functional RNA. The term “expression,” as used herein, refers to the transcription and stable accumulation of sense (mRNA) from the heterologous nucleic acid molecule described herein. Expression may also refer to translation of mRNA into a polypeptide. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cells at most times at a substantial similar level are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity. A promoter is generally bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of the polymerase.

[0032] The promoter can be heterologous to the nucleic acid molecule encoding the one or more polypeptides. The promoter can be heterologous or derived from a strain being from the same genus or species as the yeast host cell. In an embodiment, the promoter is derived from the same genus or species of the yeast host cell and the heterologous polypeptide is derived from different genus than the yeast host cell.

[0033] In the context of the present disclosure, the promoter controlling the expression of the heterologous polypeptide or the native polypeptide can be a constitutive promoter (such as, for example, tef2p (e.g., the promoter of the tef2 gene), cwp2p (e.g., the promoter of the cwp2 gene), ssal p (e.g., the promoter of the ssa1 gene), enol p (e.g., the promoter of the enol gene), hxk1 (e.g., the promoter of the hxk1 gene) and pgkl p (e.g., the promoter of the pgk1 gene). In some embodiment, the promoter is or comprises tef2p (e.g., the promoter of the tef2 gene). In some embodiment, the promoter is or comprises adhl p (e.g., the promoter of the adh1 gene). However, in some embodiments, it is preferable to limit the expression of the polypeptide. As such, the promoter controlling the expression of the heterologous polypeptide or the native polypeptide can be an inducible or modulated promoters such as, for example, a glucose-regulated promoter (e.g., the promoter of the hxt7 gene (referred to as hxt7p)) or a sulfite-regulated promoter (e.g., the promoter of the gpd2 gene (referred to as gpd2p or the promoter of the fzf1 gene (referred to as the fzf1 p)), the promoter of the ssu1 gene (referred to as ssul p), the promoter of the ssu1-r gene (referred to as ssur1-rp). In an embodiment, the promoter is an anaerobic-regulated promoters, such as, for example tdhlp (e.g., the promoter of the tdh1 gene), pau5p (e.g., the promoter of the pau5 gene), hor7p (e.g., the promoter of the hor7 gene), adhl p (e.g., the promoter of the adh1 gene), tdh2p (e.g., the promoter of the tdh2 gene), tdh3p (e.g., the promoter of the tdh3 gene), gpdl p (e.g., the promoter of the gdp1 gene), cdc19p (e.g., the promoter of the cdc19 gene), eno2p (e.g., the promoter of the eno2 gene), pdcl p (e.g., the promoter of the pdc1 gene), hxt3p (e.g., the promoter of the hxt3 gene), dan1 (e.g., the promoter of the dan1 gene) and tpil p (e.g., the promoter of the tpi1 gene). One or more promoters can be used to allow the expression of each heterologous polypeptides in the recombinant yeast host cell.

[0034] Still in the context of the present disclosure, the promoter controlling the expression of the heterologous polypeptide, or the native polypeptide can be a glycolytic promoter. For example, the glycolytic promoter can be a promoter (or a combination of promoters) from an alcohol dehydrogenase gene, a glucose-6-phosphate isomerase gene, a phosphofructokinase gene, an aldolase gene, a triosephosphate isomerase gene, a glyceraldehyde-3-phosphate dehydrogenase gene, a 3- phosphoglycerate kinase gene, a phosphoglycerate mutase, an enolase and / or a pyruvate kinase gene.

[0035] In some embodiments, the present disclosure concerns the expression of a heterologous polypeptide, a variant thereof or a fragment thereof in a host cell. A variant comprises at least one amino acid difference when compared to the amino acid sequence of the wildtype polypeptide. The polypeptide “variants” have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the corresponding wildtype heterologous polypeptides described herein. The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. The level of identity can be determined conventionally using known computer programs. Identity can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignments of the sequences disclosed herein were performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PEN ALT Y= 10). Default parameters for pairwise alignments using the Clustal method were KTUPLB 1 , GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0036] The variants exhibit the biological activity associated with the wild-type heterologous polypeptide. In an embodiment, the variant polypeptide exhibits at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the biological activity (which can be, in some embodiments, the enzymatic activity) of the corresponding wildtype heterologous polypeptide. The biological activity of the variants can be determined by methods and assays known in the art.

[0037] The variants described herein may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide for purification of the polypeptide.

[0038] A “variant” can be a conservative variant or an allelic variant. As used herein, a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the polypeptide. A substitution, insertion or deletion is said to adversely affect the protein when the altered sequence prevents or disrupts a biological function associated with the polypeptide. For example, the overall charge, structure, or hydrophobic-hydrophilic properties of the polypeptide can be altered without adversely affecting a biological activity. Accordingly, the amino acid sequence can be altered, for example to render the polypeptide more hydrophobic or hydrophilic, without adversely affecting the biological activity of the polypeptide. A “variant” can be a fragment of a heterologous wild-type polypeptide or fragment of a variant polypeptide. In some embodiments, polypeptide “fragments” have at least at least 50, 100, 200, 300, 400, 500 or more consecutive amino acids of the corresponding wildtype polypeptide or the variant. A fragment comprises at least one less amino acid residue when compared to the amino acid sequence of the corresponding wild-type heterologous polypeptide or of the variant polypeptide. In some embodiments, the fragment can comprise a N-terminal truncation of the heterologous polypeptide, and / or a C-terminal truncation of the heterologous polypeptides. In some embodiments, the fragments corresponding to the wild-type polypeptide or variant polypeptide to which the signal sequence was removed. In some embodiments, the “fragments” have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the corresponding wild-type polypeptides or variants. In some embodiments, fragments of the polypeptides can be employed for producing the corresponding full-length enzyme by peptide synthesis. Therefore, the fragments can be employed as intermediates for producing the full-length polypeptide.

[0039] In the context of the present disclosure, the fragments exhibit the biological activity of the heterologous wild-type polypeptide or of the variant polypeptide. In an embodiment, the fragment polypeptide exhibits at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the biological activity of the corresponding heterologous wild-type polypeptide or of the variant. The biological activity of fragments can be determined by methods and assays known in the art.

[0040] In some additional embodiments, the present disclosure also provides reducing the expression of or inactivating a native gene ortholog of a native gene known to encode a native polypeptide. A “gene ortholog” is understood to be a gene in a different species that evolved from a common ancestral gene by speciation. In the context of the present invention, a gene ortholog encodes a polypeptide exhibiting the same biological function than the native polypeptide.

[0041] In some further embodiments, the present disclosure also provides reducing the expression or inactivating a native gene paralog of a native gene known to encode a native polypeptide. A “gene paralog” is understood to be a gene related by duplication within the genome. In the context of the present invention, a gene paralog encodes a polypeptide that could exhibit the same biological function than the native polypeptide. The recombinant host cell of the present disclosure has the ability (which can be intrinsic and / or provided or increased by the genetic modifications introduced) to convert a biomass into one or more fermentation products. The recombinant yeast host cell can be a yeast or a fungal cell. In the context of the present disclosure, the recombinant yeast host cell is a fermenting yeast cell because it is capable of converting the biomass into the one or more fermentation products. Suitable recombinant yeast yeast host cells can be, for example, from the genus Blastobotrys (formely known as Arxula), Candida, Debaryomyces, Hanseniaspora (formely known as Kloeckera), Kazachstania, Komagataella, Kluyveromyces, Ogataea, Pichia (formely known as Hansenula), Phaffia, Saccharomyces, Scheffersomyces, Schwanniomyces, or Yarrowia. Suitable yeast species can include, for example, S. cerevisiae (including, but not limited to, var. diastaticus), Saccharomyces uvarum, Kazachstania bulderi, Kazachstania barnetti, Kazachstania exigua, Kluyveromyces lactis, Kluyveromyces marxianus, Komagataella phaffii, Candida albicans, Candida utilis, Scheffersomyces stipitis, Pichia kudriavzevii, Yarrowia lipolytica, Ogataea polymorpha, Phaffia rhodozyma, Blastobotrys adeninivorans, Debaryomyces hansenii, or Schwanniomyces polymorphus. Suitable recombinant fungal host cells can be from the genus Phaffia, Schizosaccharomyces, and include, in some embodiments, the species Schizzosaccharomyces pombe. In some embodiments, the host cell can be an oleaginous yeast cell. For example, the oleaginous yeast host cell can be from the genus Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rhodotorula, Trichosporon or Yarrowia. In some alternative embodiments, the host cell can be an oleaginous microalgae host cell (e.g., for example, from the genus Thraustochytrium or Schizochytrium). In an embodiment, the fermenting yeast or recombinant yeast host cell is from the genus Saccharomyces and, in some embodiments, from the species Saccharomyces cerevisiae.

[0042] In some embodiments, the recombinant yeast host cell of the present disclosure provides an increased fermentation kinetic when compared to a control yeast host cell comprising the first engineered metabolic pathway to convert the carbohydrate into acetyl-coA comprising a heterologous polypeptide having phosphoketolase activity as defined in b); and the second engineered metabolic pathways to convert acetyl-CoA into the fermentation product as defined in c), but lacking the upregulated pyruvate kinase as defined in a). “Fermentation kinetics” refers to the rate at which microorganisms, such as the recombinant yeast of the present disclosure, convert substrates (carbohydrates) into fermentation products. Fermentation kinetics specifically focuses on the conversion of glucose into the fermentation product and carbon dioxide by the recombinant yeast during the fermentation process until the completion of the fermentation. The skilled person of the art would be able to determine when the fermentation could be considered as completed, for example when available carbon in the fermentation medium is exhausted and / or when no activity of fermentation by the yeast is observed in the fermentation medium. Fermentation kinetics can be measured using various methodswell known in the art, including monitoring the specific gravity of the fermentation medium throughout the fermentation process, measuring the fermentation product and / or the sugars content of the fermentation medium, and / or analyzing the production of carbon dioxide during fermentation. In some embodiment, the preferred method to determine fermentation kinetic is monitoring production of carbon dioxide during fermentation. The methods to monitor carbon dioxide during fermentation are deemed to be within the scope of those skilled in the art. In some embodiment, the monitoring could be performed by automated CO2 analysis (ACAN). The ACAN consists of a custom platform for the 60mL serum bottles with tubes connected to a needle in the septum of the serum bottle, which is then connected to 0 to 10mL / min mass flow meter via multichannel HPLC valves and solenoids to allow for multiplexing the measurement of multiple vessels. A computer program controls the valves and solenoids while recording data for each bottle individually, and continually cycles between all active bottles. At the end of the fermentation the collected data is graphed by CO2 production rates (mL / min) and the integral of those rates to estimate the total CO2 produced during the fermentation (mL). In some embodiment, the fermentation kinetic of the recombinant yeast host cell is at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% increased when compared to the fermentation kinetic of a control yeast host cell comprising the first and the second engineered metabolic pathways as defined in (b) and (c) but lacking the upregulated pyruvate kinase as defined in (a).

[0043] In some embodiment, the recombinant yeast host cell of the present disclosure provides higher fermentation product yield than a control yeast host cell comprising the first and the second engineered metabolic pathways as defined in (b) and (c) but lacking the upregulated pyruvate kinase as defined in (a). In some embodiment, the recombinant yeast host cell produces at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50% or 60% higher in fermentation product yield (W / V) than a control yeast host cell comprising the first and the second engineered metabolic pathways as defined in (b) and (c) but lacking the upregulated pyruvate kinase as defined in (a). In a more particular embodiment, the fermentation product comprises ethanol, acetone, and / or isopropanol. In some embodiment, the recombinant yeast host cell exhibits an acetone yield (w / v) at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50% or 60% higher than a control yeast host cell comprising the first and the second engineered metabolic pathways as defined in (b) and (c) but lacking the upregulated pyruvate kinase as defined in (a).

[0044] In some embodiment, the recombinant yeast host cell of the present disclosure comprises upregulated enzyme. As used herein, “upregulated” means increased in activity, e.g., increase in enzymatic activity of the enzyme as compared to activity in the wild-type yeast host cell. In some embodiment, The upregulation is typically in comparison to the expression in a wild-type cell, unless specified otherwise. In the context of the present disclosure, the wild-type cell is a wild-type yeast strain. The upregulation could be done by the known methods in the art, as for example by the addition of one or more copies of a heterologous gene encoding a heterologous polypeptide in the genome of the recombinant host, increasing activity of the polypeptide encoded by the native gene by insertion or deletion of nucleic acids residues, replacement of the native promoter by a heterologous promoter more active or by modulation of a gene regulating the expression of the native gene.

[0045] In some embodiments, the recombinant yeast host cell of the present disclosure comprises a downregulated enzyme. As used herein, “downregulated” means decreased in activity, e.g., decrease in enzymatic activity of the enzyme as compared to activity in the wild-type yeast host cell. The downregulation is typically in comparison to the expression in a wild-type cell, unless specified otherwise. In the context of the present disclosure, the wild-type cell is a wild-type yeast strain. The downregulation could be done by the known methods in the art, as for example by deletion of the native gene, inactivation of the native gene by insertion or deletion of nucleic acids residues, replacement of the native promoter by a heterologous promoter less active or that does not activate under propagation and / or fermentation conditions, or by modulation of a gene regulating the expression of the native gene.

[0046] Upregulated pyruvate kinase

[0047] The recombinant yeast host cell of the present disclosure comprises an upregulated pyruvate kinase activity. In one embodiment, the upregulated kinase activity is a heterologous pyruvate kinase. As used herein, the terms "pyruvate kinase" and "PYK" are intended to include the enzymes capable of catalyzing the conversion of phosphoenolpyruvate and ADP to pyruvate and ATP in glycolysis and plays a role in regulating cell metabolism. Pyruvate kinases include those enzymes that correspond to Enzyme Commission Number 2.7.1.40. The PYK can be native or heterologous to the recombinant yeast host cell. The recombinant yeast host cell can include a native PYK and optionally in combination a heterologous PYK. In some embodiments, the PYK is of prokaryotic or eukaryotic origin. In other embodiments, the PYK can be encoded by a pyk1 gene (e.g., PYK1) or a pyk2 gene (e.g., PYK2). In some embodiments, the PYK is derived from Saccharomyces sp., and in further embodiments from Saccharomyces cerevisiae. In such embodiments, the PYK can have the amino acid sequence of SEQ ID NO: 312, 314 or 316 or be a variant of the amino acid sequence of SEQ ID NO: 312, 314 or 316 having PYK activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PYK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 311 , 313, or 315, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 312, 314 or 316 (or a variant thereof).

[0048] First engineered metabolic pathways

[0049] The recombinant yeast host cell of the present disclosure has native and / or heterologous enzymes that function in a first engineered metabolic pathway to convert carbohydrate into acetyl-coA. In some embodiment, the first engineered metabolic pathway comprises the steps of converting glucose-6-phosphate into acetyl-coenzyme A. In another embodiment, the first engineered metabolic pathway comprises the steps of converting fructose-6-phosphate into acetyl-coenzyme A. The first engineered metabolic pathway to convert carbohydrate into acetyl-coenzyme A comprises a phosphoketolase, optionally in combination with an acetate kinase, a phosphotransacetylase, and / or an acetyl-CoA synthetase. As such, the recombinant yeast host cell of the present disclosure comprises a phosphoketolase, alone or in combination with an acetate kinase, a phosphotransacetylase, and / or an acetyl-CoA synthetase. In some embodiments, the recombinant yeast host cell of the present disclosure comprises a phosphoketolase only (and therefore lacks an acetate kinase, a phosphotransacetylase, and an acetyl-CoA synthetase). In additional embodiments, the recombinant yeast host cell of the present disclosure comprises a phosphoketolase and an acetate kinase. In further embodiments, the recombinant yeast host cell of the present disclosure comprises a phosphoketolase and a phosphotransacetylase. In yet other embodiments, the recombinant yeast host cell of the present disclosure comprises a phosphoketolase, an acetate kinase, and a phosphotransacetylase. In yet other embodiments, the recombinant yeast host cell of the present disclosure comprises a phosphoketolase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase. In yet other embodiments, the recombinant yeast host cell of the present disclosure comprises a phosphoketolase and an acetyl-CoA synthetase.

[0050] As used herein, the terms "phosphoketolase" and "PHK" are intended to include the enzymes capable of converting D-xylulose 5-phosphate, D-fructose 6-phosphate, and D- sedoheptulose 7-phosphate to acetyl-phosphate. The phosphoketolase can have a singlespecificity activity (e.g., single-specificity phosphoketolase), and be only capable of converting D-xylulose 5-phosphate to D-glyceraldehyde 3-phosphate and acetylphosphate, converting D-fructose 6-phosphate to D-erythrose 4-phosphate and acetylphosphate, or converting D-sedoheptulose 7-phosphate into D-ribose 5-phosphate and acetyl-phosphate or can have a multiple-specificity / dual-specificity (e.g., multiplespecificity or dual-specificity phosphoketolase). In one embodiment, the phosphoketolase is intended to have a single-specificity and is capable of converting D-fructose 6- phosphate into acetyl-phosphate. In another embodiment, the phosphoketolase is capable of at least converting D-fructose 6-phosphate into acetyl-phosphate. In some other embodiments, the phosphoketolase is intended to have dual-specificity and is capable of converting D-xylulose 5-phosphate and D-fructose 6-phosphate to acetyl-phosphate or is intended to be multiple-specificity and is capable of converting D-xylulose 5-phosphate, D-fructose 6-phosphate, and D-sedoheptulose 7-phosphate to acetyl-phosphate. Phosphoketolases include those enzymes that correspond to Enzyme Commission Number 4.1.2.9 and 4.1.2.22. The PHK is heterologous to the recombinant yeast host cell, and the recombinant yeast host cell of the present disclosure can comprise, in some embodiments, at least two copies of a heterologous nucleic acid encoding the PHK. In some embodiments, the PHK is of prokaryotic, fungal or eukaryotic origin. In other embodiments, the PHKcan be encoded by a phk1 gene (e.g., PHK1) or a phk2 gene (e.g., PHK2). In an embodiment, the PHK is derived from Bifidobacterium sp., and in further embodiments from Bifidobacterium adolescentis. In such embodiment, the PHK can have the amino acid sequence of SEQ ID NO: 32 or be a variant of the amino acid sequence of SEQ ID NO: 32 having PHK activity. In addition, the PHK can be encoded by the nucleic acid sequence of SEQ ID NO: 31 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 32 (or a variant thereof). In some embodiments, the PHK is derived from Bifidobacterium bifidum. In such embodiment, the PHK can have the amino acid sequence of SEQ ID NO: 40 or be a variant of the amino acid sequence of SEQ ID NO: 40 having PHK activity. In addition, the PHK can be encoded by the nucleic acid sequence of SEQ ID NO: 39 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 40 (or a variant thereof). In some embodiments, the PHK can be obtained from Bifidobacterium gallicium. In such embodiment, the PHK can have the amino acid sequence of SEQ ID NO: 42 or be a variant of the amino acid sequence of SEQ ID NO: 42 having PHK activity. In addition, the PHK can be encoded by the nucleic acid sequence of SEQ ID NO: 41 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 42 (or a variant thereof). In some embodiments, the PHK can be obtained from Bifidobacterium animalis. In such embodiment, the PHK can have the amino acid sequence of SEQ ID NO: 44 or be a variant of the amino acid sequence of SEQ ID NO: 44 having PHK activity. In addition, the PHK can be encoded by the nucleic acid sequence of SEQ ID NO: 43 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 44 (or a variant thereof). In some embodiments, the PHK can be obtained from Bifidobacterium breve. In such embodiment, the PHK can have the amino acid sequence of SEQ ID NO: 64 or be a variant of the amino acid sequence of SEQ ID NO: 64 having PHK activity. In addition, the PHK can be encoded by the nucleic acid sequence of SEQ ID NO: 63 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 64 (or a variant thereof). In some embodiments, the PHK can be obtained from Bifidobacterium longum. In such embodiment, the PHK can have the amino acid sequence of SEQ ID NO: 68 or be a variant of the amino acid sequence of SEQ ID NO: 68 having PHK activity. In addition, the PHK can be encoded by the nucleic acid sequence of SEQ ID NO: 67 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 68 (or a variant thereof). In some embodiments, the PHK is derived from Bifidobacterium asteroides. In such embodiment, the PHK can have the amino acid sequence of SEQ ID NO: 344 or be a variant of the amino acid sequence of SEQ ID NO: 344 having PHK activity. In addition, the PHK can be encoded by the nucleic acid sequence of SEQ ID NO: 343 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 344 (or a variant thereof). In an embodiment, the PHK can be obtained from Lactiplantibacillus sp., and in further embodiments from Lactiplantibacillus plantarum. In such embodiment, the PHK can have the amino acid sequence of SEQ ID NO: 46 or be a variant of the amino acid sequence of SEQ ID NO: 46 having PHK activity. In addition, the PHK can be encoded by the nucleic acid sequence of SEQ ID NO: 45 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 46 (or a variant thereof). Alternatively, the PHK can have the amino acid sequence of SEQ ID NO: 60 or be a variant of the amino acid sequence of SEQ ID NO: 60 having PHK activity. In addition, the PHK can be encoded by the nucleic acid sequence of SEQ ID NO: 59 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 60 (or a variant thereof). Alternatively, the PHK can have the amino acid sequence of SEQ ID NO: 62 or be a variant of the amino acid sequence of SEQ ID NO: 62 having PHK activity. In addition, the PHK can be encoded by the nucleic acid sequence of SEQ ID NO: 61 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 62 (or a variant thereof). In some embodiments, the PHK can be obtained from Lactiplantibacillus casei. In such embodiment, the PHK can have the amino acid sequence of SEQ ID NO: 38 or be a variant of the amino acid sequence of SEQ ID NO: 38 having PHK activity. In addition, the PHK can be encoded by the nucleic acid sequence of SEQ ID NO: 37 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 38 (or a variant thereof). In some embodiments, the PHK can be obtained from Lactiplantibacillus acidophilus. In such embodiment, the PHK can have the amino acid sequence of SEQ ID NO: 48 or be a variant of the amino acid sequence of SEQ ID NO: 48 having PHK activity. In addition, the PHK can be encoded by the nucleic acid sequence of SEQ ID NO: 47 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 48 (or a variant thereof). In some embodiments, the PHK can be obtained from Lactiplantibacillus pentosus. In such embodiment, the PHK can have the amino acid sequence of SEQ ID NO: 66 or be a variant of the amino acid sequence of SEQ ID NO: 66 having PHK activity. In addition, the PHK can be encoded by the nucleic acid sequence of SEQ ID NO: 65 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 66 (or a variant thereof). In an embodiment, the PHK can be obtained from Leuconostoc sp., and in further embodiments from Leuconostoc mesenteroides. In such embodiment, the PHK can have the amino acid sequence of SEQ ID NO: 56 or be a variant of the amino acid sequence of SEQ ID NO: 56 having PHK activity. In addition, the PHK can be encoded by the nucleic acid sequence of SEQ ID NO: 55 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 56 (or a variant thereof). In an embodiment, the PHK can be obtained from Oenococcus sp., and in further embodiments from Oenococcus oeni. In such embodiment, the PHK can have the amino acid sequence of SEQ ID NO: 58 or be a variant of the amino acid sequence of SEQ ID NO: 58 having PHK activity. In addition, the PHK can be encoded by the nucleic acid sequence of SEQ ID NO: 57 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 58 (or a variant thereof). In an embodiment, the PHK can be obtained from Clostridium sp., and in further embodiments from Clostridium acetobutylicum. In such embodiment, the PHKcan have the amino acid sequence of SEQ ID NO: 70 or be a variant of the amino acid sequence of SEQ ID NO: 70 having PHK activity. In addition, the PHK can be encoded by the nucleic acid sequence of SEQ ID NO: 69 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 70 (or a variant thereof). In an embodiment, the PHK can be obtained from Aspergillus sp., and in further embodiments from Aspergillus niger. In such embodiment, the PHK can have the amino acid sequence of SEQ ID NO: 34 or be a variant of the amino acid sequence of SEQ ID NO: 34 having PHK activity. In addition, the PHK can be encoded by the nucleic acid sequence of SEQ ID NO: 33 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 34 (or a variant thereof). In some embodiments, the PHK can be obtained from Aspergillus nidulans. In such embodiment, the PHK can have the amino acid sequence of SEQ ID NO: 52 or be a variant of the amino acid sequence of SEQ ID NO: 52 having PHK activity. In addition, the PHK can be encoded by the nucleic acid sequence of SEQ ID NO: 51 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 52 (or a variant thereof). In some embodiments, the PHK can be obtained from Aspergillus clavatus. In such embodiment, the PHK can have the amino acid sequence of SEQ ID NO: 54 or be a variant of the amino acid sequence of SEQ ID NO: 54 having PHK activity. In addition, the PHK can be encoded by the nucleic acid sequence of SEQ ID NO: 53 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 54 (or a variant thereof). In an embodiment, the PHK can be obtained from Neurospora sp., and in further embodiments from Neurospora crassa. In such embodiment, the PHK can have the amino acid sequence of SEQ ID NO: 36 or be a variant of the amino acid sequence of SEQ ID NO: 36 having PHK activity. In addition, the PHK can be encoded by the nucleic acid sequence of SEQ ID NO: 35 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 36 (or a variant thereof). In an embodiment, the PHK can be obtained from Penicillium sp., and in further embodiments from Penicillium chrysogenum. In such embodiment, the PHK can have the amino acid sequence of SEQ ID NO: 50 or be a variant of the amino acid sequence of SEQ ID NO: 50 having PHK activity. In addition, the PHK can be encoded by the nucleic acid sequence of SEQ ID NO: 49 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 50 (or a variant thereof). In some embodiments, the PHK can have the amino acid sequence of any one of SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, or 70. In further embodiments, the PHK can be a variant of the amino acid sequence of SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, or 70, provided that the variant has PHK activity.

[0051] As indicated above, the recombinant yeast host cell of the present disclosure can comprise at least two copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some embodiments, the recombinant yeast host cell comprises two copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some additional embodiments, the recombinant yeast host cell of the present disclosure comprises at least three copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some embodiments, the recombinant yeast host cell comprises three copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some additional embodiments, the recombinant yeast host cell of the present disclosure comprises at least four copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some embodiments, the recombinant yeast host cell comprises four copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some additional embodiments, the recombinant yeast host cell of the present disclosure comprises at least five copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some embodiments, the recombinant yeast host cell comprises five copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some additional embodiments, the recombinant yeast host cell of the present disclosure comprises at least six copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some embodiments, the recombinant yeast host cell comprises six copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some additional embodiments, the recombinant yeast host cell of the present disclosure comprises at least seven copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some embodiments, the recombinant yeast host cell comprises seven copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some additional embodiments, the recombinant yeast host cell of the present disclosure comprises at least eight copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some embodiments, the recombinant yeast host cell comprises eight copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome.

[0052] In some embodiment, the recombinant yeast host cell of the present disclosure comprises a fusion polypeptide comprising both glucose-6-phosphate isomerase (PGI) and phosphoketolase (PHK) activities to increase the shuttling of fructose 6-P into acetyl-P. The fusion polypeptides, which can be referred as PGI-PHK fusions in the present disclosure, comprises at least two moieties, each moiety having a distinct enzymatic activity. The fusion polypeptides of the present disclosure comprise at least one moiety exhibiting glucose-6-phosphate isomerase (PGI) activity. The fusion polypeptide of the present disclosure comprises at least one moiety exhibiting phosphoketolase (PHK) activity. The fusion polypeptides of the present disclosure exhibit both glucose-6- phosphate isomerase (PGI) activity and phosphoketolase (PHK) activity. The fusion polypeptides of the present disclosure can convert glucose-6-P into acetyl-P. The at least two moieties of the fusion polypeptides can be joined using one or more covalent bonds, just as, for example, one or more amine bonds. The at least two moieties of the fusion polypeptides can be joined directly to one another. Alternatively, the at least two moieties of the fusion polypeptides can be joined indirectly to one another by the presence of a linker. In some embodiments, the linker can comprise one or more amino acid residues. The fusion polypeptides can include, at their amino end, the PGI moiety. In such embodiment, the fusion polypeptide can include, at their carboxy end, the PHK moiety. Alternatively, the fusion polypeptide can include, at their amino end, the PHK moiety. In such embodiment, the fusion polypeptide can include, at their carboxy end, the PGI moiety. Embodiments of the fusion polypeptide are described in US provisional patent application 63 / 613,290 filed on December 21 , 2023 and PCT application PCT / IB2024 / 062646 filed on December 13, 2024, and herewith incorporated in its entirety. In an embodiment, the PHK is a polypeptide which is not a moiety in a fusion polypeptide.

[0053] Glucose-6-phosphate isomerases or PGIs, also known as phosphoglucose isomerases or phosphohexose isomerases, are classified under Enzyme Commission No. 5.3.1.9 and are intended to encompass enzymes capable of converting D-glucose 6-phosphate (e.g., glucose 6-phosphate) into [3-D-fructofuranose 6-phosphate (e.g., fructose 6-phosphate). Polypeptides (including fusion polypeptides) having PGI activity have the ability of converting D-glucose 6-phosphate (e.g., glucose 6-phosphate) into [3-D-fructofuranose 6- phosphate (e.g., fructose 6-phosphate). In the context of the present disclosure, the PGI moiety of the fusion polypeptide provides the fusion polypeptide with the ability of converting D-glucose 6-phosphate (e.g., glucose 6-phosphate) into [3-D-fructofuranose 6- phosphate (e.g., fructose 6-phosphate). The PGI moiety of the fusion polypeptides can be of prokaryotic origin. In some embodiments, the PGI moiety of prokaryotic origin can be a variant of a PGI. In an embodiment, the PGI moiety can be obtained from Escherichia sp., and in further embodiments from Escherichia coli. In such embodiment, the PGI moiety can have the amino acid sequence of SEQ ID NO: 4 or be a variant of the amino acid sequence of SEQ ID NO: 4 having PGI activity. In addition, the PGI moiety can be encoded by the nucleic acid sequence of SEQ ID NO: 3 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 4 (or a variant thereof). In an embodiment, the PGI moiety can be obtained from Bifidobacterium sp., and in further embodiments from Bifidobacterium adolescentis. In such embodiment, the PGI moiety can have the amino acid sequence of SEQ ID NO: 16 or be a variant of the amino acid sequence of SEQ ID NO: 16 having PGI activity. In addition, the PGI moiety can be encoded by the nucleic acid sequence of SEQ ID NO: 15 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 16 (or a variant thereof). In an embodiment, the PGI moiety can be obtained from Bacillus sp., and in further embodiments from Bacillus subtilis. In such embodiment, the PGI moiety can have the amino acid sequence of SEQ ID NO: 26 or be a variant of the amino acid sequence of SEQ ID NO: 26 having PGI activity. In addition, the PGI moiety can be encoded by the nucleic acid sequence of SEQ ID NO: 25 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 26 (or a variant thereof). In an embodiment, the PGI moiety can be obtained from Lachnoclostridium sp., and in further embodiments from Lachnoclostridium phytofermentans. In such embodiment, the PGI moiety can have the amino acid sequence of SEQ ID NO: 28 or be a variant of the amino acid sequence of SEQ ID NO: 28 having PGI activity. In addition, the PGI moiety can be encoded by the nucleic acid sequence of SEQ ID NO: 27 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 28 (or a variant thereof). In an embodiment, the PGI moiety can be obtained from Lactiplantibacillus sp., and in further embodiments from Lactiplantibacillus plantarum. In such embodiment, the PGI moiety can have the amino acid sequence of SEQ ID NO: 30 or be a variant of the amino acid sequence of SEQ ID NO: 30 having PGI activity. In addition, the PGI moiety can be encoded by the nucleic acid sequence of SEQ ID NO: 29 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 30 (or a variant thereof). The PGI moiety of the fusion polypeptides can be of eukaryotic origin. In some embodiments, the PGI moiety of eukaryotic origin can be a variant of a PGI. In some additional embodiments, the PGI moiety can be of fungal origin. In an embodiment, the PGI moiety can be obtained from Saccharomyces sp., and in further embodiments from Saccharomyces cerevisiae. In such embodiment, the PGI moiety can have the amino acid sequence of SEQ ID NO: 2 or be a variant of the amino acid sequence of SEQ ID NO: 2 having PGI activity. In addition, the PGI moiety can be encoded by the nucleic acid sequence of SEQ ID NO: 1 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2 (or a variant thereof). In an embodiment, the PGI moiety can be obtained from Kluyveromyces sp., and in further embodiments from Kluyveromyces marxianus. In such embodiment, the PGI moiety can have the amino acid sequence of SEQ ID NO: 6 or be a variant of the amino acid sequence of SEQ ID NO: 6 having PGI activity. In addition, the PGI moiety can be encoded by the nucleic acid sequence of SEQ ID NO: 5 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 6 (or a variant thereof). In another embodiment, the PGI moiety can be obtained from Kluyveromyces lactis. In such embodiment, the PGI moiety can have the amino acid sequence of SEQ ID NO: 8 or be a variant of the amino acid sequence of SEQ ID NO: 8 having PGI activity. In addition, the PGI moiety can be encoded by the nucleic acid sequence of SEQ ID NO: 7 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 8 (or a variant thereof). In an embodiment, the PGI moiety can be obtained from Ogataea sp., and in further embodiments from Ogataea parapolymorpha. In such embodiment, the PGI moiety can have the amino acid sequence of SEQ ID NO: 10 or be a variant of the amino acid sequence of SEQ ID NO: 10 having PGI activity. In addition, the PGI moiety can be encoded by the nucleic acid sequence of SEQ ID NO: 9 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 10 (ora variant thereof). In an embodiment, the PGI moiety can be obtained from Zygosaccharomyces sp., and in further embodiments from Zygosaccharomyces rouxii. In such embodiment, the PGI moiety can have the amino acid sequence of SEQ ID NO: 12 or be a variant of the amino acid sequence of SEQ ID NO: 12 having PGI activity. In addition, the PGI moiety can be encoded by the nucleic acid sequence of SEQ ID NO: 11 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 12 (or a variant thereof). In an embodiment, the PGI moiety can be obtained from Candida sp., and in further embodiments from Candida albicans. In such embodiment, the PGI moiety can have the amino acid sequence of SEQ ID NO: 14 or be a variant of the amino acid sequence of SEQ ID NO: 14 having PGI activity. In addition, the PGI moiety can be encoded by the nucleic acid sequence of SEQ ID NO: 13 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 14 (or a variant thereof). In further embodiments, the PGI moiety can be obtained from Candida boidinii. In such embodiment, the PGI moiety can have the amino acid sequence of SEQ ID NO: 24 or be a variant of the amino acid sequence of SEQ ID NO: 24 having PGI activity. In addition, the PGI moiety can be encoded by the nucleic acid sequence of SEQ ID NO: 23 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 24 (or a variant thereof). In an embodiment, the PGI moiety can be obtained from Millerozyma sp., and in further embodiments from Millerozyma farinosa. In such embodiment, the PGI moiety can have the amino acid sequence of SEQ ID NO: 18 or be a variant of the amino acid sequence of SEQ ID NO: 18 having PGI activity. In addition, the PGI moiety can be encoded by the nucleic acid sequence of SEQ ID NO: 17 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 18 (or a variant thereof). In an embodiment, the PGI moiety can be obtained from Meyerozyma sp., and in further embodiments from Meyerozyma guilliermondii. In such embodiment, the PGI moiety can have the amino acid sequence of SEQ ID NO: 20 or be a variant of the amino acid sequence of SEQ ID NO: 20 having PGI activity. In addition, the PGI moiety can be encoded by the nucleic acid sequence of SEQ ID NO: 19 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 20 (or a variant thereof). In an embodiment, the PGI moiety can be obtained from Scheffersomyces sp., and in further embodiments from Scheffersomyces stipitis. In such embodiment, the PGI moiety can have the amino acid sequence of SEQ ID NO: 22 or be a variant of the amino acid sequence of SEQ ID NO: 22 having PGI activity. In addition, the PGI moiety can be encoded by the nucleic acid sequence of SEQ ID NO: 21 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 22 (or a variant thereof). In some embodiments, the PGI moiety can have the amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30. In further embodiments, the PGI can be a variant of the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30, provided that the variant has PGI activity. In some embodiments, the N-terminal methionine residue which is included in the amino acid sequence of the PGI moiety is removed upon inclusion in the fusion protein. In the context of the present disclosure, the PHK moiety of the fusion polypeptide provides the fusion polypeptide with the ability of converting D-fructose 6-phosphate into acetylphosphate. In one embodiment, the PHK moiety of the fusion polypeptide provides the fusion polypeptide with the ability of converting D-xylulose 5-phosphate and D-fructose 6- phosphate into acetyl-phosphate. In some embodiments, the PHK moiety can have the amino acid sequence of any one of SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, or 70. In further embodiments, the PHK can be a variant of the amino acid sequence of SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, or 70, provided that the variant has PHK activity. In some embodiments, the N-terminal methionine residue which is included in the amino acid sequence of the PHK moiety is removed upon inclusion in the fusion protein.

[0054] In additional embodiments of the present disclosure, there may be a linker covalently associated to both moieties. In some embodiments, the linker may be an amino acid linker. In such embodiment, the amino terminus of the linker is covalently associated to the carboxy terminus of one of the moieties and the carboxy terminus of the linker is covalently associated with the amino terminus of the other moiety. In an embodiment, the amino terminus of the linker is associated (directly or indirectly) with the carboxy terminus of the PGI moiety, while the carboxy terminus of the linker is associated (directly or indirectly) with the amino terminus of the PHK moiety. In other embodiments, the amino terminus of the linker is associated (directly or indirectly) with the carboxy terminus of the PHK moiety, while the carboxy terminus of the linker is associated (directly or indirectly) with the amino terminus of the PGI moiety. The amino acid linker comprises at least one amino acid residue, and in some embodiments a plurality of consecutive amino acid residues. In some additional embodiments, the linker can comprise between one and fifty (50) amino acid residues. In some embodiments, the amino acid linker can be rigid (and can have for example the amino acid sequence of SEQ ID NO: 72 or a variant thereof or be encoded by the nucleic acid sequence of SEQ ID NO: 71 or a degenerate sequence thereof encoding the amino acid sequence of SEQ ID NO: 72). In additional embodiments, the amino acid linker can be flexible (and can have for example the amino acid sequence of SEQ ID NO: 74 or a variant thereof or be encoded by the nucleic acid sequence of SEQ ID NO: 73 or a degenerate sequence thereof encoding the amino acid sequence of SEQ ID NO: 74). Additional amino acid linkers include, without limitations, (G)n, (GS)n; (GGS)n; (GGGS)n; (GGGGS)n; (GGSG)n; (GSAT)n, wherein n = is an integer between 1 to 8 (or more). In an embodiment, the amino acid linker L is (GGGGS)n (also referred to as G4S) and, in still further embodiments, the amino acid linker L comprises more than one G4S (SEQ ID NO: 309) motifs. The amino acid linker can also be, in some embodiments, GSAGSAAGSGEF (SEQ ID NO: 310). Further amino acid linkers include, without limitations, (EAAK)n and (EAAAK)n, wherein n = is an integer between 1 to 8 (or more). In some embodiments, the one or more (EAAK)n / (EAAAK)n motifs can be separated by one or more additional amino acid residues. Additional amino acid linkers include those having one or more (AP)n motifs wherein n = is an integer between 1 to 10 (or more).

[0055] In some embodiments, the recombinant yeast host cell of the present disclosure comprises glucose-6-phosphate isomerase (PGI) not provided as a moiety of the fusion polypeptide, and phosphoketolase (PHK) not provided as a moiety of the fusion polypeptide, the activities of which increase the shuttling of fructose 6-P into acetyl-P. PGI not provided as a moiety of the fusion polypeptide and PHK not provided as a moiety of the fusion polypeptide are expressed as separate polypeptides.

[0056] In an embodiment, the recombinant yeast host cell can convert glucose-6-phosphate to acetyl-coA and / or fructose-6-phosphate to acetyl-coA. As indicated on Figure 1 , once glucose-6-phosphate and / or fructose-6-phosphate are converted to acetyl-P by the fusion polypeptide (or a variant thereof) and / or by the phosphoketolase (or a variant thereof, including a PHK not provided as a moiety of the fusion polypeptide), acetyl-P can be further converted into acetyl-coenzyme A via two metabolic routes. The first metabolic route is the enzymatic conversion of acetyl-P directly into acetyl-coA using a polypeptide having phosphotransacetylase activity. The second metabolic route relies on the successive enzymatic conversion of acetyl-P into acetate (by the enzymatic activity of a polypeptide having acetate kinase activity or a polypeptide having glycerol-3-P phosphatase activity) and the conversion of acetate into acetyl-coA (by the enzymatic activity of a polypeptide having acetyl-coenzyme A synthetase activity.

[0057] The first engineered metabolic pathway to convert carbohydrate into acetyl-coenzyme A can include, in some embodiments, a phosphotransacetylase. As such, the recombinant yeast host cell of the present disclosure can include, in such embodiments, a phosphotransacetylase. As used herein, the terms "phosphotransacetylase" and "PTA" are intended to include the enzymes capable of converting acetyl-phosphate into acetyl- coA. Phosphotransacetylases include those enzymes that correspond to Enzyme Commission Number 2.3.1.8. The PTA can be native or heterologous to the recombinant yeast host cell. In some embodiments, the PTA is of prokaryotic or eukaryotic origin. In other embodiments, the PTA can be encoded by a pta gene (e.g., PTA). In some embodiments, the PTA is derived from Bifidobacterium sp., and in further embodiments from Bifidobacterium adolescentis. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 76 or be a variant of the amino acid sequence of SEQ ID NO: 76 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 75, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 76 (a variant thereof). In further embodiments, the PTA is derived from Bifidobacterium animalis. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 98 or be a variant of the amino acid sequence of SEQ ID NO: 98 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 97, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 98 (a variant thereof). In some further embodiments, the PTA is derived from Bifidobacterium bifidum. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 160 or be a variant of the amino acid sequence of SEQ ID NO: 160 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 159, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 160 (a variant thereof). In some embodiments, the PTA is derived from Leuconostoc sp., and in further embodiments from Leuconostoc mesenteroides. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 78 or be a variant of the amino acid sequence of SEQ ID NO: 78 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 77, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 78 (a variant thereof). In some embodiments, the PTA is derived from Oenococcus sp., and in further embodiments from Oenococcus oenii. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 80 or be a variant of the amino acid sequence of SEQ ID NO: 80 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 79, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 80 (a variant thereof). In some embodiments, the PTA is derived from Salmonella sp., and in further embodiments from Salmonella enterica. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 88 or 90 or be a variant of the amino acid sequence of SEQ ID NO: 88 or 90 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 87 or 89, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 88 or 90 (a variant thereof). In some embodiments, the PTA is derived from Bacillus sp., and in further embodiments from Bacillus subtilis. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 86 or be a variant of the amino acid sequence of SEQ ID NO: 86 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 85, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 86 (a variant thereof). In some embodiments, the PTA is derived from Clostridium sp., and in further embodiments from Clostridium kluveryi. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 92 or be a variant of the amino acid sequence of SEQ ID NO: 92 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 91 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 92 (a variant thereof). In some embodiments, the PTA is derived from Phytophthora sp., and in further embodiments from Phytophthora ramorum. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 94 or be a variant of the amino acid sequence of SEQ ID NO: 94 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 93, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 94 (a variant thereof). In some embodiments, the PTA is derived from Globisporangium sp., and in further embodiments from Globisporangium splendens. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 96 or be a variant of the amino acid sequence of SEQ ID NO: 96 having PTA activity. The recombinant yeast host cell can include, a nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 95, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 96 (a variant thereof). In some embodiments, the PTA is derived from Clostridium sp., and in further embodiments from Clostridium phytofermentans. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 100 or be a variant of the amino acid sequence of SEQ ID NO: 100 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 99, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 100 (a variant thereof). In some embodiments, the PTA is derived from Holophagae sp., and in further embodiments from Holophagae bacterium. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 102 or be a variant of the amino acid sequence of SEQ ID NO: 102 having PTA activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 101 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ I D NO: 102 (a variant thereof). In some embodiments, the PTA is derived from Azetobacter sp., and in further embodiments from Aztobacter vinelandii. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 82 or be a variant of the amino acid sequence of SEQ ID NO: 82 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 81 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 82 (a variant thereof). In some embodiments, the PTA is derived from Lactobacillus sp., and in further embodiments from Lactiplantibacillus plantarum. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 84 or be a variant of the amino acid sequence of SEQ ID NO: 84 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 83, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 84 (a variant thereof). Additional sources of PTA that can be included in the recombinant yeast host cell of the present disclosure include, without limitation, Phytophthora sp., (including but not limited to Phytophthora cactorum, Phytophthora parasitica, or Phytophthora idaei), Chlamydomonas sp. (including but not limited to Chlamydomonas reinhardtii), Clostridium sp. (including but not limited to Clostridium cellulolyticum), and Microcystis sp. (including but not limited to Microcystis aeruginosa).

[0058] The first engineered metabolic pathway to convert carbohydrate into acetyl-coenzyme A can include, in some embodiments, an acetate kinase. As such, the recombinant yeast host cell of the present disclosure can include, in such embodiments, an acetate kinase. As used herein, the terms "acetate kinase" and "ACK" are intended to include the enzymes capable of converting acetate into acetyl-phosphate (acetyl-P). Acetate kinases include those enzymes that correspond to Enzyme Commission Number 2.72.1. The ACK can be native or heterologous to the recombinant yeast host cell. In some embodiments, the ACK is of prokaryotic or eukaryotic origin. In other embodiments, the ACK can be encoded by an ack gene (e.g., ACK). In some embodiments, the ACK is derived from Bifidobacterium sp., and in further embodiments from Bifidobacterium adolescentis. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 108 or be a variant of the amino acid sequence of SEQ ID NO: 108 having ACK activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 107, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 108 (a variant thereof). In some embodiments, the ACK is derived from Leuconostoc sp., and in further embodiments from Leuconostoc mesenteroides. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 110 or be a variant of the amino acid sequence of SEQ ID NO: 110 having ACK activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 109, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 110 (a variant thereof). In some embodiments, the ACK is derived from Oenococcus sp., and in further embodiments from Oenococcus oenii. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 112 or be a variant of the amino acid sequence of SEQ ID NO: 112 having ACK activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 111 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 112 (a variant thereof). In some embodiments, the ACK is derived from Escherichia sp., and in further embodiments from Escherichia coli. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 114 or be a variant of the amino acid sequence of SEQ ID NO: 114 having ACK activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 113, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 114 (a variant thereof). In some embodiments, the ACK is derived from Bacillus sp., and in further embodiments from Bacillus subtilis. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 116 or be a variant of the amino acid sequence of SEQ ID NO: 116 having ACK activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 115, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 116 (a variant thereof). In some embodiments, the ACK is derived from Clostridium sp., and in further embodiments from Clostridium acetobutylicum. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 118 or be a variant of the amino acid sequence of SEQ ID NO: 118 having ACK activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 117, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 118 (a variant thereof). In some embodiments, the ACK is derived from Salmonella sp., and in further embodiments from Salmonella enterica. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 120 or be a variant of the amino acid sequence of SEQ ID NO: 120 having ACK activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 119, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 120 (a variant thereof). In some embodiments, the ACK is derived from Phytophthora sp., and in further embodiments from Phytophthora ramorum. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 122 or be a variant of the amino acid sequence of SEQ ID NO: 122 having ACK activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 121 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 122 (a variant thereof). In some embodiments, the ACK is derived from Chlamydomonas sp., and in further embodiments from Chlamydomonas reinhardtii. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 124 or be a variant of the amino acid sequence of SEQ ID NO: 124 having ACK activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 123, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 124 (a variant thereof). In some embodiments, the ACK is derived from Aspergillus sp., and in further embodiments from Aspergillus nidulans. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 126 or be a variant of the amino acid sequence of SEQ ID NO: 126 having ACK activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 125, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 126 (a variant thereof). In some embodiments, the ACK is derived from Lactiplantibacillus sp., and in further embodiments from Lactiplantibacillus plantarum. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 128 or be a variant of the amino acid sequence of SEQ ID NO: 128 having ACK activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 127, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 128 (a variant thereof). In some embodiments, the ACK is derived from Clostridium sp., and in further embodiments from Clostridium kluveryi. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 130 or be a variant of the amino acid sequence of SEQ ID NO: 130 having ACK activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 129, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 130 (a variant thereof).

[0059] The first engineered metabolic pathway to convert carbohydrate into acetyl-coenzyme A can include, in some embodiments, an acetyl-coA synthetase. As such, the recombinant yeast host cell of the present disclosure can include, in some embodiments, an acetylcoenzyme A synthetase. As used herein, the term “acetyl-coA synthetase” and “ACS” are intended to include the enzymes capable of converting acetate into acetyl-coA. Acetyl-coA synthetases include those enzymes that correspond to Enzyme Commission Number 6.2.1.1. The ACS can be native or heterologous to the recombinant yeast host cell. The ACS can be of prokaryotic or eukaryotic origin. In other embodiments, the ACS can be encoded by an acs1 gene (e.g., ACS1) or an acs2 gene (e.g., ACS2). In some embodiments, the ACS is derived from Saccharomyces sp., and in further embodiments from Saccharomyces cerevisiae. In such embodiments, the ACS can have the amino acid sequence of SEQ ID NO: 132 or 134 or be a variant of the amino acid sequence of SEQ ID NO: 132 or 134 having ACS activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ACS. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 131 or 133 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 132 or 134 (a variant thereof). In some embodiments, the ACS is derived from Zygosaccharomyces sp., and in further embodiments from Zygosaccharomyces bailii. In such embodiments, the ACS can have the amino acid sequence of SEQ ID NO: 136 or be a variant of the amino acid sequence of SEQ ID NO: 136 having ACS activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACS. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 135 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 136 (a variant thereof). In some embodiments, the ACS is derived from Salmonella sp., and in further embodiments from Salmonella enterica. In such embodiments, the ACS can have the amino acid sequence of SEQ ID NO: 138 or be a variant of the amino acid sequence of SEQ ID NO: 138 having ACS activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACS. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ I D NO: 137 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 138 (a variant thereof). In some embodiments, the ACS is derived from Acetobacter sp., and in further embodiments from Acetobacter aceti. In such embodiments, the ACS can have the amino acid sequence of SEQ ID NO: 140 or be a variant of the amino acid sequence of SEQ ID NO: 140 having ACS activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACS. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ I D NO: 139 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 140 (a variant thereof). In some embodiments, the ACS is derived from Escherichia sp., and in further embodiments from Escherichia coli. In such embodiments, the ACS can have the amino acid sequence of SEQ ID NO: 142 or be a variant of the amino acid sequence of SEQ ID NO: 142 having ACS activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACS. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 141 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 142 (a variant thereof). Additional sources of ACS that can be included in the recombinant yeast host cell of the present disclosure include, without limitation, Aedes togoi, Aliivibrio fischeri, Amaranth us sp., Arabidopsis thaliana, Archaeoglobus fulgidus, Aspergillus nidulans, Aspergillus niger, Bacillus subtilis, Bos taurus, Bradyrhizobium japonicum, Cereibacter sphaeroides, Cryptosporidium parvum, Dunaliella tertiolecta, Euglena gracilis, Haloarcula marismortui, Homo sapiens, Hordeum vulgare, Ignicoccus hospitalis, Marmota monax, Methanosarcina acetivorans, Methanosarcina sp., Methanothermobacter thermautotrophicus, Methanothrix soehngenii, Methanothrix thermoacetophila (Uniprot A0B8F1 for exemple), Moorella thermoacetica, Mus musculus, Mus musculus, Oryctolagus cuniculus, Ovis aries, Pelotomaculum thermopropionicum, Penicillium chrysogenum, Phycomyces blakesleeanus, Pinus radiata, Pisum sativum, Populus trichocarpa, Pseudomonas putida (Uniprot Q6EMJ3 for example), Pyrobaculum aerophilum, Pyrococcus furiosus, Rattus norvegicus, Rhodotorula diobovata, Roseovarius sp., Saccharopolyspora erythraea, Spinacia oleracea, Streptomyces lividans, Taxus sp., and Zea mays.

[0060] In specific embodiments, the recombinant yeast host cell of the present disclosure comprises a native or heterologous enzyme that function in the first engineered metabolic pathway to convert acetate into acetyl-coA. In such embodiments, the native or heterologous enzyme comprises an activated, upregulated or overexpressed acetyl-coA synthetase 2 (ACS2). In some embodiments, the ACS2 is derived from Saccharomyces sp., and in further embodiments from Saccharomyces cerevisiae. In such embodiments, the ACS can have the amino acid sequence of SEQ ID NO: 132 or be a variant of the amino acid sequence of SEQ ID NO: 132 having ACS activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACS. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 131 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 132 (a variant thereof).

[0061] The recombinant yeast host cell of the present disclosure can include, in some embodiments, a glycerol-3-phosphate phosphatase. As used herein, the term “glycerol-3- phosphate phosphatase” or “GPP” are intended to include the enzymes capable of dephosphorylating, amongst other substrates, acetyl-phosphate to generate acetate. Glycerol-3-phosphate phosphatases include those enzymes that correspond to Enzyme Commission Number 3.1.3.21. The GPP can be native or heterologous to the recombinant yeast host cell. The GPP can be of prokaryotic or eukaryotic origin. In some embodiments, the GPP can be encoded by a gpp1 gene (e.g., GPP1) or a gpp2 gene (e.g., GPP2). In some embodiments, the GPP is derived from Saccharomyces sp., and in further embodiments from Saccharomyces cerevisiae. In such embodiments, the GPP can have the amino acid sequence of SEQ ID NO: 104 or 106 or be a variant of the amino acid sequence of SEQ ID NO: 104 or 106 having GPP activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the GPP. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 103 or 105 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 104 or 106 (a variant thereof). In some embodiments, the GPP1 is derived from Arabidopsis sp., and in further embodiments, from Arabidopsis thaliana (and can be associated with the NCBI Gene ID: 828690 or a variant thereof). In some embodiments, the GPP1 is derived from Lachnellula sp., and in further embodiments, from Lachnellula hyalina (and can be associated with the NCBI Gene ID: 41983435 or a variant thereof). In some embodiments, the GPP1 is derived from Scheffersomyces sp., and in further embodiments, from Scheffersomyces stipitis (and can be associated with the NCBI Gene ID: 4836794 or a variant thereof). In some embodiments, the GPP1 is derived from Aspergillus sp., and in further embodiments, from Aspergillus melleus (and can be associated with the NCBI Gene ID: 70114293 or a variant thereof). In some embodiments, the GPP1 is derived from Didymosphaeria sp., and in further embodiments, from Didymosphaeria variabile (and can be associated with the NCBI Gene ID: 80909868 or a variant thereof). In some embodiments, the GPP1 is derived from Ophidiomyces sp., and in further embodiments, from Ophidiomyces ophidiicola (and can be associated with the NCBI Gene ID: 73310376 or a variant thereof). In some embodiments, the GPP1 is derived from Purpureocillium sp., and in further embodiments, from Purpureocillium takamizusanense (and can be associated with the NCBI Gene ID: 72063146 or a variant thereof). In some embodiments, the GPP1 is derived from Chlorella sp., and in further embodiments, from Chlorella variabilis (and can be associated with the NCBI Gene ID: 17352997 or a variant thereof). In some embodiments, the GPP2 is derived from Colletotrichum sp., and in further embodiments, from Colletotrichum aenigma (and can be associated with the NCBI Gene ID: 59244509 or a variant thereof), from Colletotrichum aenigma (and can be associated with the NCBI Gene ID: 59249666 or a variant thereof), or from Colletotrichum siamense (and can be associated with the NCBI Gene ID: 59275989 or a variant thereof). In some embodiments, the GPP2 is derived from a parvovirus and, in some embodiments, from the Artibeusjamaicensis parvovirus 1 (and can be associated with the NCBI Gene ID: 11605582 or a variant thereof), or the Eidolon helvum parvovirus (and can be associated with the NCBI Gene ID: 11763499 or a variant thereof). In some embodiments, the GPP2 is derived from Saccharomyces sp., and in further embodiments, from Saccharomyces paradoxus (and can be associated with the NCBI Gene ID: 54630122 or a variant thereof). In some embodiments, the GPP2 is derived from Nicotiana sp., and in further embodiments, from Nicotiana attenuate (and can be associated with the NCBI Gene ID: 109234217 or a variant thereof). In some embodiments, the GPP2 is derived from Arabidopsis sp., and in further embodiments, from Arabidopsis thaliana (and can be associated with the NCBI Gene ID: 835849 or a variant thereof). In some embodiments, the GPP2 is derived from Sugiyamaella sp., and in further embodiments, from Sugiyamaella lignohabitans (and can be associated with the NCBI Gene ID: 30035078 or a variant thereof).

[0062] Second engineered metabolic pathways

[0063] The recombinant yeast host cell of the present disclosure includes a native and / or heterologous enzyme that functions in a second engineered metabolic pathway to convert acetyl-coenzyme A (acetyl-CoA) into a fermentation product. In specific embodiments, the fermentation product is acetone, isopropanol, and / or ethanol. In a further embodiment, the second engineered metabolic pathway concerns the conversion of acetyl-coenzyme A into acetone and / or isopropanol. In such embodiment, the engineered metabolic pathway to convert acetyl-coenzyme A into acetone and / or isopropanol comprises at least a thiolase, a coenzyme A transferase, and an acetoacetate decarboxylase. In such embodiment, the engineered pathway also convert acetate along with acetyl-coenzyme A. In another embodiment, the engineered metabolic pathway to convert acetyl-coenzyme A into acetone comprise at least a thiolase, a HMG CoA synthase, a HMG CoA lyase, and an acetoacetate decarboxylase. In another embodiment, the engineered metabolic pathway to convert acetyl-coenzyme A to acetone comprise at least a thiolase, acetoacetyl-CoA hydrolase, and an acetoacetate decarboxylase.

[0064] In some embodiments, the engineered metabolic pathway to convert acetyl-coenzyme A and acetate to acetone comprises a thiolase. As used herein, the terms "thiolase", “THL”, “ERG 10” and "PHAA" are intended to include the enzymes capable of converting acetyl- coA into acetoacetyl-coA. Thiolases include enzymes that correspond to Enzyme Commission Number 2.3.1.9. The thiolase can be native or heterologous to the recombinant yeast host cell. The recombinant yeast host cell can include a native thiolase and optionally in combination a heterologous thiolase. In some embodiments, the thiolase is of prokaryotic or eukaryotic origin. In other embodiments, the thiolase can be encoded by a th I gene (e.g., THL), an erg10 gene (e.g., ERG10), or a phaA gene (e.g., PHAA). In some embodiments, the thiolase is derived from Saccharomyces sp., and in further embodiments from Saccharomyces cerevisiae. In such embodiments, the thiolase can have the amino acid sequence of SEQ ID NO: 144 or be a variant of the amino acid sequence of SEQ ID NO: 144 having thiolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the thiolase. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 143, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 144 (a variant thereof). In some embodiments, the thiolase is derived from Clostridium sp., and in further embodiments from Clostridium acetobutylicum. In such embodiments, the thiolase can have the amino acid sequence of SEQ ID NO: 162 or be a variant of the amino acid sequence of SEQ ID NO: 162 having thiolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 162 having thiolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the thiolase. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 161 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 162 (a variant thereof). In some embodiments, the thiolase is from Clostridium kluyveri. In such embodiments, the thiolase can have the amino acid sequence of SEQ ID NO: 164 or be a variant of the amino acid sequence of SEQ ID NO: 164 having thiolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the thiolase. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 163, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 164 (a variant thereof). In some embodiments, the thiolase is derived from Clostridium beijerinckii. In such embodiments, the thiolase can have the amino acid sequence of SEQ ID NO: 180 or be a variant of the amino acid sequence of SEQ ID NO: 180 having thiolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the thiolase. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 179, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ I D NO: 180 (a variant thereof). In some embodiments, the thiolase is derived from Cupriavidus sp., and in further embodiments from Cupriavidus necator. In such embodiments, the thiolase can have the amino acid sequence of SEQ ID NO: 166 or be a variant of the amino acid sequence of SEQ ID NO: 166 having thiolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the thiolase. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 165, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 166 (a variant thereof). In some embodiments, the thiolase is derived from Yarrowia sp., and in further embodiments from Yarrowia lipolytica. In such embodiments, the thiolase can have the amino acid sequence of SEQ ID NO: 168 or be a variant of the amino acid sequence of SEQ ID NO: 168 having thiolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the thiolase. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 167, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 168 (a variant thereof). In some embodiments, the thiolase is derived from Thermoanaerobacterium sp., and in further embodiments from Thermoanaerobacterium thermosaccharolyticum. In such embodiments, the thiolase can have the amino acid sequence of SEQ ID NO: 170 or be a variant of the amino acid sequence of SEQ ID NO: 170 having thiolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the thiolase. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 169, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 170 (a variant thereof). In some embodiments, the thiolase is derived from Saccogiossus sp., and in further embodiments from Saccogiossus kowalevskii. In such embodiments, the thiolase can have the amino acid sequence of SEQ ID NO: 172 or be a variant of the amino acid sequence of SEQ ID NO: 172 having thiolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the thiolase. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 171 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 172 (a variant thereof thereof). In some embodiments, the thiolase is derived from Strongylocentrotus sp., and in further embodiments from Strongylocentrotus purpuratus. In such embodiments, the thiolase can have the amino acid sequence of SEQ ID NO: 174 or be a variant of the amino acid sequence of SEQ ID NO: 174 having thiolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the thiolase. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 173, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 174 (a variant thereof). In some embodiments, the thiolase is derived from Paenibacillus sp., and in further embodiments from Paenibacillus polymyxa. In such embodiments, the thiolase can have the amino acid sequence of SEQ ID NO: 176 or be a variant of the amino acid sequence of SEQ ID NO: 176 having thiolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the thiolase. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 175, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 176 (a variant thereof). In some embodiments, the thiolase is derived from Zygosaccharomyces sp., and in further embodiments from Zygosaccharomyces bailii. In such embodiments, the thiolase can have the amino acid sequence of SEQ ID NO: 178 or be a variant of the amino acid sequence of SEQ ID NO: 178 having thiolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the thiolase. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 177, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 178 (a variant thereof). Additional sources of thiolases that can be included in the recombinant yeast host cell include, without limitation, Arabidopsis thaliana (Uniprot Q8S4Y1 or Q9FIK7, for example), Aspergillus fumigatus (Uniprot B0XMC1 , for example), Bacillus subtilis, Bacopa monnieri (Uniprot D9U856, for example), Bos taurus, Bradyrhizobium japonicum, Candida tropicalis, Catharanthus roseus, Caulobacter vibrioides, Clonorchis sinensis (Uniprot G7YHN5, for example), Dictyostelium discoideum (Uniprot Q86AD9, for example), Enterococcus faecalis, Escherichia coli (Uniprot P76461 , for example), Euphorbia helioscopia (Uniprot A0A0M4F9H9, for example), Gallus gallus (Uniprot F1 NT20, for example), Ginkgo biloba (Uniprot A0A1S6KJS1 , for example), Halobacterium sp., Haloferax mediterranei (Uniprot I3R3D1 , I3R3D0, I3RA72, or l3RA71 , for example), Helianthus annuus (Uniprot D2IH11 , for example), Homo sapiens (Uniprot Q9BWD1 , for example), Medicago sativa (Uniprot D0EUY6, for example), Metallosphaera sedula (Uniprot A4YEH9, for example), Methanothermococcus thermolithotrophicus (Uniprot A0A384E138, for example), Mycolicibacterium smegmatis, Ostrinia scapulalis (Uniprot B7XEI5, for example), Pyricularia oryzae, Pyrobaculum neutrophilum (Uniprot B1YB71 , for example), Rattus norvegicus, Rhizobium sp., Sanghuangporus baumii, Thermus thermophilus, Vitis vinifera x Vitis riparia, and Zoogloea ramigera (Uniprot P07256 or P07097, for example).

[0065] In some embodiments, the engineered metabolic pathway to convert acetyl-coenzyme A and acetate to acetone comprises a coenzyme A transferase. As used herein, the terms "coenzyme A transferase", “coA transferase”, “CFTA / CTFB”, and “ATOA / ATOD” are intended to include the enzymes (or enzyme moieties) capable of converting acetoacetyl- coA and acetate into acetyl-coA and acetoacetate. Coenzyme A transferases include enzymes that correspond to Enzyme Commission Number 2.8.3.8. The coA transferase can be a monomer or a dimer (as for example CTFA / CTFB). The coA transferase can be native or heterologous to the recombinant yeast host cell. The recombinant yeast host cell can include a native coA transferase and optionally in combination a heterologous coA transferase. In some embodiments, the coA transferase is of prokaryotic or eukaryotic origin. In other embodiments, the coA transferase can be encoded by the ctfa and ctfb genes (e.g., CTFA / CTFB), or by the atoA and atoD genes (e.g., ATOA / ATOD). In some embodiments, the coA transferase is derived from Alkaliphilus sp., and in further embodiments from Alkaliphilus metalliredigens. In such embodiments, the coA transferase can include a first polypeptide (CTFA) having the amino acid sequence of SEQ ID NO: 146 or being a variant of the amino acid sequence of SEQ ID NO: 146 having coA transferase activity (in the presence of CTFB). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFA. In such embodiment, the heterologous nucleic acid molecule encoding this first polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 145, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 146 (a variant thereof). Alternatively, or in combination, the coA transferase can include a second polypeptide (CTFB) having the amino acid sequence of SEQ I D NO: 148 or being a variant of the amino acid sequence of SEQ ID NO: 148 having coA transferase activity (in the presence of CTFA). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFB. In such embodiment, the heterologous nucleic acid molecule encoding this second polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 147, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 148 (a variant thereof). In some embodiments, the coA transferase is derived from Clostridium sp., and in further embodiments from Clostridium acetobutylicum. In such embodiments, the coA transferase can include a first polypeptide (CTFA) having the amino acid sequence of SEQ ID NO: 182 or being a variant of the amino acid sequence of SEQ ID NO: 182 having coA transferase activity (in the presence of CTFB). The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the CTFA. In such embodiment, the heterologous nucleic acid molecule encoding this first polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 181 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 182 (a variant thereof). Alternatively, or in combination, the coA transferase can include a second polypeptide (CTFB) having the amino acid sequence of SEQ ID NO: 184 or being a variant of the amino acid sequence of SEQ ID NO: 184 having coA transferase activity (in the presence of CTFA). The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the CTFB. In such embodiment, the heterologous nucleic acid molecule encoding this second polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 183, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 184 (a variant thereof). In some embodiments, the coA transferase is derived from Thermosipho sp., and in further embodiments from Thermosipho melanesiensis. In such embodiments, the coA transferase can include a first polypeptide (CTFA) having the amino acid sequence of SEQ ID NO: 186 or being a variant of the amino acid sequence of SEQ ID NO: 186 having coA transferase activity (in the presence of CTFB). The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the CTFA. In such embodiment, the heterologous nucleic acid molecule encoding this first polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 185, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 186 (a variant thereof). Alternatively, or in combination, the coA transferase can include a second polypeptide (CTFB) having the amino acid sequence of SEQ ID NO: 188 or being a variant of the amino acid sequence of SEQ ID NO: 188 having coA transferase activity (in the presence of CTFA). The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the CTFB. In such embodiment, the heterologous nucleic acid molecule encoding this second polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 187, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 188 (a variant thereof). In some embodiments, the coA transferase is derived from Escherichia sp., and in further embodiments from Escherichia coli. In such embodiments, the coA transferase can include a first polypeptide (CTFA) having the amino acid sequence of SEQ ID NO: 190 or being a variant of the amino acid sequence of SEQ ID NO: 190 having coA transferase activity (in the presence of CTFB). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFA. In such embodiment, the heterologous nucleic acid molecule encoding this first polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 189, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 190 (a variant thereof). Alternatively, or in combination, the coA transferase can include a second polypeptide (CTFB) having the amino acid sequence of SEQ I D NO: 192 or being a variant of the amino acid sequence of SEQ ID NO: 192 having coA transferase activity (in the presence of CTFA). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFB. In such embodiment, the heterologous nucleic acid molecule encoding this second polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 191 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ I D NO: 192 (a variant thereof). In some embodiments, the coA transferase is derived from Paenibacillus sp., and in further embodiments from Paenibacillus polymyxa. In such embodiments, the coA transferase can include a first polypeptide (CTFA) having the amino acid sequence of SEQ ID NO: 194 or being a variant of the amino acid sequence of SEQ ID NO: 194 having coA transferase activity (in the presence of CTFB). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFA. In such embodiment, the heterologous nucleic acid molecule encoding this first polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 193, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 194 (a variant thereof). Alternatively, or in combination, the coA transferase can include a second polypeptide (CTFB) having the amino acid sequence of SEQ ID NO: 196 or being a variant of the amino acid sequence of SEQ ID NO: 196 having coA transferase activity (in the presence of CTFA). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFB. In such embodiment, the heterologous nucleic acid molecule encoding this second polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 195, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 196 (a variant thereof). In some embodiments, the coA transferase is derived from Clostridium sp., and in further embodiments from Clostridium beijerinckii. In such embodiments, the coA transferase can include a first polypeptide (CTFA) having the amino acid sequence of SEQ ID NO: 198 or being a variant of the amino acid sequence of SEQ ID NO: 198 having coA transferase activity (in the presence of CTFB). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFA. In such embodiment, the heterologous nucleic acid molecule encoding this first polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 197, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 198 (a variant thereof). Alternatively, or in combination, the coA transferase can include a second polypeptide (CTFB) having the amino acid sequence of SEQ ID NO: 200 or being a variant of the amino acid sequence of SEQ ID NO: 200 having coA transferase activity (in the presence of CTFA). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFB. In such embodiment, the heterologous nucleic acid molecule encoding this second polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 199, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 200 (a variant thereof). In some embodiments, the coA transferase is derived from Clostridium saccharoperbutylacetonicum. In such embodiments, the coA transferase can include a first polypeptide (CTFA) having the amino acid sequence of SEQ ID NO: 202 or being a variant of the amino acid sequence of SEQ ID NO: 202 having coA transferase activity (in the presence of CTFB). The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the CTFA. In such embodiment, the heterologous nucleic acid molecule encoding this first polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 201 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 202 (a variant thereof). Alternatively, or in combination, the coA transferase can include a second polypeptide (CTFB) having the amino acid sequence of SEQ ID NO: 204 or being a variant of the amino acid sequence of SEQ ID NO: 204 having coA transferase activity (in the presence of CTFA). The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the CTFB. In such embodiment, the heterologous nucleic acid molecule encoding this second polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 203, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 204 (a variant thereof). In some embodiments, the coA transferase is derived from Clostridium sp., and in further embodiments from Clostridium sticklandii. In such embodiments, the coA transferase can include a first polypeptide (CTFA) having the amino acid sequence of SEQ ID NO: 206 or being a variant of the amino acid sequence of SEQ ID NO: 206 having coA transferase activity (in the presence of CTFB). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFA. In such embodiment, the heterologous nucleic acid molecule encoding this first polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 205, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 206 (a variant thereof). Alternatively, or in combination, the coA transferase can include a second polypeptide (CTFB) having the amino acid sequence of SEQ I D NO: 208 or being a variant of the amino acid sequence of SEQ ID NO: 208 having coA transferase activity (in the presence of CTFA). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFB. In such embodiment, the heterologous nucleic acid molecule encoding this second polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 207, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 208 (a variant thereof). In some embodiments, the coA transferase is derived from Brevibacillus sp., and in further embodiments from Brevibacillus laterosporus. In such embodiments, the coA transferase can include a first polypeptide (CTFA) having the amino acid sequence of SEQ ID NO: 210 or being a variant of the amino acid sequence of SEQ ID NO: 210 having coA transferase activity (in the presence of CTFB). The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the CTFA. In such embodiment, the heterologous nucleic acid molecule encoding this first polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 209, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 210 (a variant thereof). Alternatively, or in combination, the coA transferase can include a second polypeptide (CTFB) having the amino acid sequence of SEQ ID NO: 212 or being a variant of the amino acid sequence of SEQ ID NO: 212 having coA transferase activity (in the presence of CTFA). The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the CTFB. In such embodiment, the heterologous nucleic acid molecule encoding this second polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 211 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 212 (a variant thereof). In some embodiments, the coA transferase is derived from Clostridium sp., and in further embodiments from Clostridium bovifaecis. In such embodiments, the coA transferase can include a first polypeptide (CTFA) having the amino acid sequence of SEQ ID NO: 214 or being a variant of the amino acid sequence of SEQ ID NO: 214 having coA transferase activity (in the presence of CTFB). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFA. In such embodiment, the heterologous nucleic acid molecule encoding this first polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 213, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 214 (a variant thereof). Alternatively, or in combination, the coA transferase can include a second polypeptide (CTFB) having the amino acid sequence of SEQ ID NO: 216 or being a variant of the amino acid sequence of SEQ ID NO: 216 having coA transferase activity (in the presence of CTFA). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFB. In such embodiment, the heterologous nucleic acid molecule encoding this second polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 215, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 216 (a variant thereof). Additional sources of coA transferase that can be included in the recombinant yeast host cell include, without limitation, Acetobacter aceti (Uniprot B3EY95, for example), Anaerobutyricum hallii (Uniprot Q2QIH1 or D2WEY8, for example), Anaerostipes caccae (Uniprot Q2QB27 or B0MC58, for example), Butyricicoccus porcorum, Butyrivibrio fibrisolvens (Uniprot D2WEY7, for example), Coprococcus sp., Faecalibacterium prausnitzii (Uniprot Q2QIH0, A8SFP6, C7H5K4, or D2WEZ2, for example), Megasphaera elsdenii, Roseburia hominis (Uniprot Q2TME9, for example), Roseburia intestinalis (Uniprot C7GB37, for example), Roseburia inulinivorans (Uniprot D2WEY6, for example), Thermoanaerobacterium saccharolyticum, Trypanosoma brucei, Eubacterium nodatum, and Eubacterium rectale (Uniprot D2WEY1 , for example).

[0066] In some embodiments, the engineered metabolic pathway to convert acetyl-coenzyme A and acetate into acetone comprises an acetoacetate decarboxylase. As used herein, the terms “acetoacetate decarboxylase”, and “ADC” are intended to include the enzymes capable of converting acetoacetate into acetone and carbon dioxide. Acetoacetate decarboxylases include enzymes that correspond to Enzyme Commission Number 4.1.1.4. The acetoacetate decarboxylase can be native or heterologous to the recombinant yeast host cell. The recombinant yeast host cell can include a native acetoacetate decarboxylase and optionally in combination a heterologous acetoacetate decarboxylase. In some embodiments, acetoacetate decarboxylase is of prokaryotic or eukaryotic origin. In other embodiments, the acetoacetate decarboxylase can be encoded by an adc gene (e.g., ADC). In some embodiments, the acetoacetate decarboxylase is derived from Paenibacillus sp., and in further embodiments from Paenibacillus polymyxa. In such embodiments, the acetoacetate decarboxylase can include a polypeptide having the amino acid sequence of SEQ ID NO: 150 or 218 or be a variant of the amino acid sequence of SEQ ID NO: 150 or 218 having ADC activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 149 or 217, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 150 or 218 (a variant thereof). In some embodiments, the acetoacetate decarboxylase is derived from Clostridium sp., and in further embodiments from Clostridium acetobutylicum. In such embodiments, the acetoacetate decarboxylase can include a polypeptide having the amino acid sequence of SEQ ID NO: 220 or be a variant of the amino acid sequence of SEQ ID NO: 220 having ADC activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ADC. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 219, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 220 (a variant thereof). In some embodiments, the acetoacetate decarboxylase is derived from Clostridium beijerinckii. In such embodiments, the acetoacetate decarboxylase can include a polypeptide having the amino acid sequence of SEQ ID NO: 222 or be a variant of the amino acid sequence of SEQ I D NO: 222 having ADC activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ADC. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 221 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 222 (a variant thereof). In some embodiments, the acetoacetate decarboxylase is derived from Bacillus sp., and in further embodiments from Bacillus amyloliquefaciens. In such embodiments acetoacetate decarboxylase can include a polypeptide having the amino acid sequence of SEQ ID NO: 236 or be a variant of the amino acid sequence of SEQ I D NO: 236 having ADC activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ADC. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 235, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 236 (a variant thereof). In some embodiments, the acetoacetate decarboxylase is derived from Rhizobium sp., and in further embodiments from Rhizobium leguminosarum. In such embodiment, the acetoacetate decarboxylase can include a polypeptide having the amino acid sequence of SEQ ID NO: 224 or be a variant of the amino acid sequence of SEQ ID NO: 224 having ADC activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ADC. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 223, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 224 (a variant thereof). In some embodiments, the acetoacetate decarboxylase is derived from Bradyrhizobium sp., and in further embodiments from Bradyrhizobium japonicum. In such embodiments, the acetoacetate decarboxylase can include a polypeptide having the amino acid sequence of SEQ ID NO: 226 or be a variant of the amino acid sequence of SEQ ID NO: 226 having ADC activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ADC. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 225, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 226 (a variant thereof). In some embodiments, the acetoacetate decarboxylase is derived from Tetrahymena sp., and in further embodiments from Tetrahymena thermophila. In such embodiments, the acetoacetate decarboxylase can include a polypeptide having the amino acid sequence of SEQ ID NO: 238 or be a variant of the amino acid sequence of SEQ ID NO: 238 having ADC activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ADC. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 237, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 238 (a variant thereof). In some embodiments, the acetoacetate decarboxylase is derived from Aspergillus sp., and in further embodiments from Aspergillus niger. In such embodiments, the acetoacetate decarboxylase can include a polypeptide having the amino acid sequence of SEQ ID NO: 228 or be a variant of the amino acid sequence of SEQ ID NO: 228 having ADC activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ADC. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 227, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 228 (a variant thereof). In some embodiments, the acetoacetate decarboxylase is derived from Lacticaseibacillus sp., and in further embodiments from Lacticaseibacillus casei. In such embodiments, the acetoacetate decarboxylase can include a polypeptide having the amino acid sequence of SEQ ID NO: 230 or be a variant of the amino acid sequence of SEQ ID NO: 230 having ADC activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ADC. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 229, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 230 (a variant thereof). In some embodiments, the acetoacetate decarboxylase is derived from Lacticaseibacillus sp., and in further embodiments from Lacticaseibacillus rhamnosus. In such embodiments, the acetoacetate decarboxylase can include a polypeptide having the amino acid sequence of SEQ ID NO: 232 or be a variant of the amino acid sequence of SEQ ID NO: 232 having ADC activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ADC. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 231 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 232 (a variant thereof). In some embodiments, the acetoacetate decarboxylase is derived from Aspergillus sp., and in further embodiments from Aspergillus bertholletiae. In such embodiments, the acetoacetate decarboxylase can include a polypeptide having the amino acid sequence of SEQ ID NO: 234 or be a variant of the amino acid sequence of SEQ ID NO: 234 having ADC activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ADC. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 233, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 234 (a variant thereof). Additional sources of ADC that can be included in the recombinant yeast host cell include, without limitation, Chromobacterium violaceum (Uniprot Q7NSA6, for example), Pseudomonas putida, and Ruminiclostridium cellulolyticum.

[0067] In some embodiments, the engineered metabolic pathway to convert acetyl-coenzyme A and acetate into acetone comprises an HMG CoA Synthase (HGMS). As used herein, the terms “HMG CoA Synthase”, and “HMGS” are intended to include the enzymes capable of converting acetoacetyl-CoA and acetyl-coA into (S)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). HMG CoA Synthase include enzymes that correspond to Enzyme Commission Number 2.3.3.10. The HMG CoA Synthase can be native or heterologous to the recombinant yeast host cell. The recombinant yeast host cell can include a native HMG CoA Synthase and optionally in combination a heterologous HMG CoA Synthase. In some embodiments, HMG CoA Synthase is of prokaryotic or eukaryotic origin. In other embodiments, the HMG CoA Synthase can be encoded by an hgms gene. In some embodiments, the HMG CoA synthase is derived from Saccharomyces sp., and in further embodiments from Saccharomyces cerevisiae. In such embodiments, the HMG CoA synthase can include a polypeptide having the amino acid sequence of SEQ ID NO: 240 or be a variant of the amino acid sequence of SEQ ID NO: 240 having HMGS activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 239, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 240 (a variant thereof). In some embodiments, the HMG CoA synthase is derived from Lacticaseibacillus sp., and in further embodiments from Lacticaseibacillus casei. In such embodiments, the HMG CoA synthase can include a polypeptide having the amino acid sequence of SEQ ID NO: 242 or be a variant of the amino acid sequence of SEQ ID NO: 242 having HMGS. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 241 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 242 (a variant thereof). In some embodiments, the HMG CoA synthase is derived from Enterococcus sp., and in further embodiments from Enterococcus faecalis. In such embodiments, the HMG CoA synthase can include a polypeptide having the amino acid sequence of SEQ ID NO: 244 or be a variant of the amino acid sequence of SEQ ID NO: 244 having HMGS activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 243, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 244 (a variant thereof). In some embodiments, the HMG CoA synthase is derived from Haloferax sp., and in further embodiments from Haloferax volcanii. In such embodiments, the HMG CoA synthase can include a polypeptide having the amino acid sequence of SEQ ID NO: 246 or be a variant of the amino acid sequence of SEQ ID NO: 246 having HMGS activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 245, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 246 (a variant thereof). In some embodiments, the HMG CoA synthase is derived from Alloscardovia sp., and in further embodiments from Alloscardovia theropitheci. In such embodiments, the HMG CoA synthase can include a polypeptide having the amino acid sequence of SEQ ID NO: 248 or be a variant of the amino acid sequence of SEQ ID NO: 248 having HMGS activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 247, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 248 (a variant thereof). In some embodiments, the HMG CoA synthase is derived from Listeria sp., and in further embodiments from Listeria monocytogenes. In such embodiments, the HMG CoA synthase can include a polypeptide having the amino acid sequence of SEQ ID NO: 250 or be a variant of the amino acid sequence of SEQ ID NO: 250 having HMGS activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 249, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 250 (a variant thereof).

[0068] In some embodiments, the engineered metabolic pathway to convert acetyl-coenzyme A and acetate into acetone comprises an HMG CoA lyaselyase (HMGCL). As used herein, the terms “HMG CoA lyaselyase”, and “HMGCL” are intended to include the enzymes capable of converting HMG-CoA into acetyl-CoA and acetoacetate. HMG CoA lyase includes enzymes that correspond to Enzyme Commission Number 4.1.3.4. The HMG CoA lyase can be native or heterologous to the recombinant yeast host cell. The recombinant yeast host cell can include a native HMG CoA lyase and optionally in combination a heterologous HMG CoA lyase. In some embodiments, HMG CoA lyas is of prokaryotic or eukaryotic origin. In other embodiments, the HMG CoA lyas can be encoded by an hmgcl gene. In some embodiments, the HMG CoA lyase is derived from Danio sp., and in further embodiments from Danio rerio. In such embodiments, the HMG CoA lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 252 or be a variant of the amino acid sequence of SEQ ID NO: 252 having HMGCL activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 251 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 252 (a variant thereof). In some embodiments, the HMG CoA lyase is derived from Pseudomonas sp., and in further embodiments from Pseudomonas monteilii. In such embodiments, the HMG CoA lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 254 or be a variant of the amino acid sequence of SEQ ID NO: 254 having HMGCL activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 253, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 254 (a variant thereof). In some embodiments, the HMG CoA lyase is derived from Pseudomonas sp., and in further embodiments from Pseudomonas wayambapalatensis. In such embodiments, the HMG CoA lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 256 or be a variant of the amino acid sequence of SEQ ID NO: 256 having HMGCL activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 255, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 256 (a variant thereof). In some embodiments, the HMG CoA lyase is derived from Azotobacter sp., and in further embodiments from Azotobacter vinelandii. In such embodiments, the HMG CoA lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 258 or be a variant of the amino acid sequence of SEQ ID NO: 258 having HMGCL activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 257, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 258 (a variant thereof). In some embodiments, the HMG CoA lyase is derived from Pseudomonas sp., and in further embodiments from Pseudomonas citronellolis. In such embodiments, the HMG CoA lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 260 or be a variant of the amino acid sequence of SEQ ID NO: 260 having HMGCL activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 259, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 260 (a variant thereof). In some embodiments, the HMG CoA lyase is derived from Pseudomonas sp., and in further embodiments from Pseudomonas cremoris. In such embodiments, the HMG CoA lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 262 or be a variant of the amino acid sequence of SEQ ID NO: 262 having HMGCL activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 261 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 262 (a variant thereof). In some embodiments, the HMG CoA lyase is derived from Pseudomonas sp., and in further embodiments from Pseudomonas chengduensis. In such embodiments, the HMG CoA lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 264 or be a variant of the amino acid sequence of SEQ ID NO: 264 having HMGCL activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 263, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 264 (a variant thereof). In some embodiments, the HMG CoA lyase is derived from Pseudomonas sp., and in further embodiments from Pseudomonas aeruginosa. In such embodiments, the HMG CoA lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 266 or be a variant of the amino acid sequence of SEQ ID NO: 266 having HMGCL activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 265, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 266 (a variant thereof). In some embodiments, the HMG CoA lyase is derived from Bacillus sp., and in further embodiments from Bacillus subtilis. In such embodiments, the HMG CoA lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 268 or be a variant of the amino acid sequence of SEQ ID NO: 268 having HMGCL activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 267, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 268 (a variant thereof). In some embodiments, the HMG CoA lyase is derived from Desulfotomaculum sp., and in further embodiments from Desulfotomaculum arcticum. In such embodiments, the HMG CoA lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 270 or be a variant of the amino acid sequence of SEQ ID NO: 270 having HMGCL activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 269, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 270 (a variant thereof). In some embodiments, the HMG CoA lyase is derived from Desulfoscipio sp., and in further embodiments from Desulfoscipio geothermicus. In such embodiments, the HMG CoA lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 272 or be a variant of the amino acid sequence of SEQ ID NO: 272 having HMGCL activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 271 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 272 (a variant thereof) In some embodiments, the HMG CoA lyase is derived from Acinetobacter sp., and in further embodiments from Acinetobacter baumannii. In such embodiments, the HMG CoA lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 274 or be a variant of the amino acid sequence of SEQ ID NO: 274 having HMGCL activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 273, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 274 (a variant thereof). In some embodiments, the HMG CoA lyase is derived from Acinetobacter sp., and in further embodiments from Acinetobacter Iwoffii. In such embodiments, the HMG CoA lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 276 or be a variant of the amino acid sequence of SEQ ID NO: 276 having HMGCL activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 275, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 276 (a variant thereof). In some embodiments, the HMG CoA lyase is derived from Moraxella sp., and in further embodiments from Moraxella caviae. In such embodiments, the HMG CoA lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 278 or be a variant of the amino acid sequence of SEQ ID NO: 278 having HMGCL activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 277, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 278 (a variant thereof). In some embodiments, the HMG CoA lyase is derived from Alcaligenaceae sp., and in further embodiments from Alcaligenaceae bacterium. In such embodiments, the HMG CoA lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 280 or be a variant of the amino acid sequence of SEQ ID NO: 280 having HMGCL activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 279, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 280 (a variant thereof). In some embodiments, the HMG CoA lyase is derived from Macaca sp., and in further embodiments from Macaca fascicularis. In such embodiments, the HMG CoA lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 282 or be a variant of the amino acid sequence of SEQ ID NO: 282 having HMGCL activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 281 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 282 (a variant thereof). In some embodiments, the HMG CoA lyase is derived from Arabidopsis sp., and in further embodiments from Arabidopsis thaliana. In such embodiments, the HMG CoA lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 284 or be a variant of the amino acid sequence of SEQ ID NO: 284 having HMGCL activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 283, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 284 (a variant thereof). In some embodiments, the HMG CoA lyase is derived from Gallus sp., and in further embodiments from Gallus gallus. In such embodiments, the HMG CoA lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 286 or be a variant of the amino acid sequence of SEQ ID NO: 286 having HMGCL activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 285, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 286 (a variant thereof).

[0069] In some embodiments, the engineered metabolic pathway to convert acetyl-coenzyme A and acetate into acetone comprises an acetoacetyl-CoA hydrolase. As used herein, the terms “acetoacetyl-CoA hydrolase”, “TESB”, “YCIA”, “FADM1”, “FADM2” and “FADM” are intended to include the enzymes capable of converting acetoacetyl-CoA into acetyl-coA and acetoacetate. Acetoacetyl-CoA hydrolase includes enzymes that correspond to Enzyme Commission Number EC 3.1.2.11. The acetoacetyl-CoA hydrolase can be native or heterologous to the recombinant yeast host cell. The recombinant yeast host cell can include a native acetoacetyl-CoA hydrolase and optionally in combination a heterologous acetoacetyl-CoA hydrolase. In some embodiments, acetoacetyl-CoA hydrolase is of prokaryotic or eukaryotic origin. In other embodiments, the acetoacetyl-CoA hydrolase can be encoded by a tesB”, “yciA”, “fadM1”, “fadM2” or “fadM gene. In some embodiments, the acetoacetyl-CoA hydrolase is derived from Escherichia sp., and in further embodiments from Escherichia coli. In such embodiments, the acetoacetyl-CoA hydrolase (TESB) can include a polypeptide having the amino acid sequence of SEQ ID NO: 287 or be a variant of the amino acid sequence of SEQ ID NO: 287 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 287 (a variant thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Escherichia sp., and in further embodiments from Escherichia coli. In such embodiments, the acetoacetyl-CoA hydrolase (YCIA) can include a polypeptide having the amino acid sequence of SEQ ID NO: 298 or be a variant of the amino acid sequence of SEQ ID NO: 298 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 298 (a variant thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Escherichia sp., and in further embodiments from Escherichia coli. In such embodiments, the acetoacetyl-CoA hydrolase (YIGI) can include a polypeptide having the amino acid sequence of SEQ ID NO: 299 or be a variant of the amino acid sequence of SEQ ID NO: 299 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 299 (a variant thereof). In some embodiments, the acetoacetyl- CoA hydrolase is derived from Escherichia sp., and in further embodiments from Escherichia coli. In such embodiments, the acetoacetyl-CoA hydrolase (YDIL) can include a polypeptide having the amino acid sequence of SEQ ID NO: 300 or be a variant of the amino acid sequence of SEQ ID NO: 300 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 300 (a variant thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Methylorubrum sp., and in further embodiments from Methylorubrum extorquens. In such embodiments, the acetoacetyl-CoA hydrolase can include a polypeptide having the amino acid sequence of SEQ ID NO: 288 or be a variant of the amino acid sequence of SEQ ID NO: 288 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 288 (a variant thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Pseudomonas sp., and in further embodiments from Pseudomonas aeruginosa. In such embodiments, the acetoacetyl-CoA hydrolase can include a polypeptide having the amino acid sequence of SEQ ID NO: 289 or be a variant of the amino acid sequence of SEQ ID NO: 289 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 289 (a variant thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Campylobacter sp., and in further embodiments from Campylobacter jejuni. In such embodiments, the acetoacetyl- CoA hydrolase can include a polypeptide having the amino acid sequence of SEQ ID NO: 290 or be a variant of the amino acid sequence of SEQ ID NO: 290 having acetoacetyl- CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 290 (a variant thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Mycobacterium sp., and in further embodiments from Mycobacterium tuberculosis. In such embodiments, the acetoacetyl-CoA hydrolase can include a polypeptide having the amino acid sequence of SEQ ID NO: 291 or be a variant of the amino acid sequence of SEQ ID NO: 291 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 291 (a variant thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Pseudomonas sp., and in further embodiments from Pseudomonas putida. In such embodiments, the acetoacetyl-CoA hydrolase can include a polypeptide having the amino acid sequence of SEQ ID NO: 292 or be a variant of the amino acid sequence of SEQ ID NO: 292 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 292 (a variant thereof). In some embodiments, the acetoacetyl- CoA hydrolase is derived from Fibrobacter sp., and in further embodiments from Fibrobacter succinogenes. In such embodiments, the acetoacetyl-CoA hydrolase can include a polypeptide having the amino acid sequence of SEQ ID NO: 293 or be a variant of the amino acid sequence of SEQ ID NO: 293 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 293 (a variant thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Alcanivorax sp., and in further embodiments from Alcanivorax borkumensis. In such embodiments, the acetoacetyl-CoA hydrolase can include a polypeptide having the amino acid sequence of SEQ ID NO: 294 or be a variant of the amino acid sequence of SEQ ID NO: 294 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 294 (a variant thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Haemophilus sp., and in further embodiments from Haemophilus influenzae. In such embodiments, the acetoacetyl-CoA hydrolase can include a polypeptide having the amino acid sequence of SEQ ID NO: 295 or be a variant of the amino acid sequence of SEQ ID NO: 295 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 295 (a variant thereof). In some embodiments, the acetoacetyl- CoA hydrolase is derived from Zymomonas sp., and in further embodiments from Zymomonas mobilis subsp. mobilis. In such embodiments, the acetoacetyl-CoA hydrolase can include a polypeptide having the amino acid sequence of SEQ ID NO: 296 or be a variant of the amino acid sequence of SEQ ID NO: 296 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 296 (a variant thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Campylobacter sp., and in further embodiments from Campylobacter jejuni. In such embodiments, the acetoacetyl-CoA hydrolase can include a polypeptide having the amino acid sequence of SEQ ID NO: 297 or be a variant of the amino acid sequence of SEQ ID NO: 297 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 297 (a variant thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Prevotella sp., and in further embodiments from Prevotella ruminicola 23. In such embodiments, the acetoacetyl-CoA hydrolase (FadM1) can include a polypeptide having the amino acid sequence of SEQ ID NO: 301 or be a variant of the amino acid sequence of SEQ ID NO: 301 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 301 (a variant thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Prevotella sp., and in further embodiments from Prevotella ruminicola. In such embodiments, the acetoacetyl-CoA hydrolase (FadM2) can include a polypeptide having the amino acid sequence of SEQ ID NO: 302 or be a variant of the amino acid sequence of SEQ ID NO: 302 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 302 (a variant thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Providencia sp., and in further embodiments from Providencia sneebia. In such embodiments, the acetoacetyl- CoA hydrolase can include a polypeptide having the amino acid sequence of SEQ ID NO: 303 or be a variant of the amino acid sequence of SEQ ID NO: 303 having acetoacetyl- CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 303 (a variant thereof).

[0070] In embodiments in which it is preferred that the recombinant yeast host cell produces more acetone and less isopropanol, the recombinant yeast host cell can be engineered to have a downregulated native alcohol dehydrogenase (ADH) gene in combination with a first heterologous alcohol dehydrogenase (ADH) gene, wherein the native ADH is capable of converting acetone into isopropanol and the first heterologous alcohol dehydrogenase has a decreased activity in the conversion of the acetone to isopropanol when compared to the native ADH intended for downregulation in the recombinant yeast host cell.

[0071] In embodiments in which it is preferred that the recombinant yeast host cell produces more acetone and less isopropanol, the recombinant yeast host cell can be engineered to have a downregulated native alcohol dehydrogenase (ADH) gene, wherein the native ADH is capable of converting acetone into isopropanol. As used herein, the terms “alcohol dehydrogenase” or “ADH” is intended to include the enzymes capable of converting aldehyde and / or ketone into an alcohol. ADH includes enzymes that correspond to Enzyme Commission Number 1.1.1.1. The term "downregulated" means decreased in activity, e.g., decrease in enzymatic activity of the enzyme as compared to activity in a native host organism. As used in the context of the present disclosure, the downregulation of the native adh gene refers to a genetic modification which limits or impedes the expression of the native adh gene, when compared to a corresponding yeast strain which does not bear such genetic modification. In some instances, the downregulation reduces but still allows the expression of the native adh gene and / or the expression of the native ADH. In other instances, the downregulation inhibits the expression of the native adh gene and / or the expression of the native ADH. The downregulation could be done by the known methods in the art, as for example by deletion of the native adh gene, inactivation of the adh gene by insertion or deletion of nucleic acids residues, replacement of the native promoter by a heterologous promoter less active or that does not activate under propagation and / or fermentation conditions, or by modulation of a gene regulating the expression of the native adh gene. In one embodiment, the genetic modification for downregulating the native adh gene is a deletion of the native adh gene. In one embodiment the native ADH is the native ADH that is mainly responsible for the conversion of the acetone to isopropanol by the yeast host cell. In another embodiment, the native ADH is ADH1 (encoded by the adh1 gene). In another embodiment, the native ADH1 is encoded by the native adh1 gene from S. cerevisiae. Embodiments of such downregulation of the adh gene are disclosed in US provisional patent application 63 / 595,169, filed on November 1 , 2023 and incorporated herewith in its entirety.

[0072] In embodiments in which the recombinant yeast host cell has a downregulated native alcohol dehydrogenase (ADH) gene, the recombinant yeast host cell also comprises a first heterologous ADH. The first heterologous alcohol dehydrogenase has a decreased activity in the conversion of the acetone to isopropanol when compared to the native ADH intended for downregulation in the recombinant yeast host cell. In one embodiment, the first heterologous alcohol dehydrogenase has a secondary alcohol activity inferior to the secondary alcohol activity of the native ADH intended for the downregulation in the yeast host cell. In another embodiment, the first heterologous ADH allows the recombinant yeast host cell comprising (a) an engineered metabolic pathway to convert acetyl-CoA and acetate into acetone and (b) a downregulated ADH to produce a yield of isopropanol inferior to 0.5 g / L in a YPD media containing 120 g / L glucose at pH 6.0 following 48 hours of fermentation. In one embodiment, the first heterologous ADH is a bacterial ADH. In other embodiments, the first heterologous ADH can be encoded by an adha, adhp, oradh4 gene. In a further embodiment, the first heterologous ADH is not a S. cerevisiae ADH 1 or is not encoded by a S. cerevisiae adh1 gene. In some embodiments, the first heterologous ADH is derived from Zymomonas sp., and in further embodiments from Zymomonas mobilis. In such embodiments, the first heterologous ADH (ADHA) can include a polypeptide having the amino acid sequence of SEQ ID NO: 304 or be a variant of the amino acid sequence of SEQ ID NO: 304 having ADH activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the first heterologous ADH. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 304 (a variant thereof). In some embodiments, the first heterologous ADH is derived from Zymomonas sp., and in further embodiments from Zymomonas mobilis. In such embodiments, the first heterologous ADH (ADHP) can include a polypeptide having the amino acid sequence of SEQ ID NO: 305 or be a variant of the amino acid sequence of SEQ ID NO: 305 having ADH activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the first heterologous ADH. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 305 (a variant thereof). In some embodiments, the first heterologous ADH is derived from Saccharomyces sp., and in further embodiments from Saccharomyces cerevisiae. In such embodiments, the first heterologous ADH (ADH4) can include a polypeptide having the amino acid sequence of SEQ ID NO: 306 or be a variant of the amino acid sequence of SEQ ID NO: 306 having ADH activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the first heterologous ADH. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 306 (a variant thereof). Embodiments of such first heterologous adh gene are disclosed in US provisional patent application 63 / 595,169, filed on November 1 , 2023 and incorporated herewith in its entirety.

[0073] In embodiments in which it is preferred that the recombinant yeast host cell produces more isopropanol and less acetone, the recombinant yeast host cell can be engineered to upregulated alcohol dehydrogenase (ADH) activity. In such embodiment, the upregulated ADH is capable of converting acetone into isopropanol. As used herein, the terms “ADH” is intended to include the enzymes capable of converting aldehyde and / or ketone into an alcohol. ADH includes enzymes that correspond to Enzyme Commission Number 1.1.1.1. The upregulated ADH can be native and / or heterologous to the recombinant yeast host cell. The recombinant yeast host cell can include a native ADH and optionally in combination a second heterologous ADH. In some embodiments, the second heterologous ADH is of prokaryotic or eukaryotic origin. In other embodiments, the second heterologous ADH can be encoded by an adh gene.

[0074] Additional genetic modifications

[0075] To provide additional substrate to the first engineered pathways, the recombinant yeast host cell of the present disclosure includes / expresses a native and / or heterologous enzyme that functions in the engineered non-oxidative pentose phosphate pathway. In such embodiment, the recombinant yeast host cell of the present disclosure comprises at least one of: a transaldolase, a transketolase, an epimerase, or an isomerase. In some embodiments, the transaldolase has the ability to convert glyceraldehyde 3-phosphate into erythrose 4-phosphate and / or has the ability to convert sedoheptulose 7-phosphate into fructose 6-phosphate. In some embodiments, the transaldolase is native or heterologous to the recombinant yeast host cell. The transaldolase can be native or heterologous. In some embodiments, the recombinant yeast host cell comprises both native and heterologous copies of one or more gene encoding the transaldolase. In additional embodiments, the transaldolase is of prokaryotic or eukaryotic origin. In some embodiments, the transaldolase is a TAL1 polypeptide which is encoded by a tall gene. In some additional embodiments, the transaldolase is derived from Saccharomyces sp.; and in further embodiments, is derived from Saccharomyces cerevisiae. In such embodiment, the transaldolase can have the amino acid sequence of SEQ ID NO: 154 (a variant thereof); and / or be encoded by a heterologous nucleic acid molecule comprising the nucleic acid molecule of SEQ ID NO: 153 or be a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 154 (or a variant thereof). In some embodiments, the transketolase has the ability to convert ribose 5-phosphate into glyceraldehyde 3-phosphate, to convert fructose 6-phosphate into xylulose 5-phosphate; to convert xylulose 5-phoshate into sedoheptulose 7-phosphate; and / or to convert glyceraldehyde 3-phosphate into erythrose 4-phosphate. The transketolase can be native or heterologous. In some embodiments, the recombinant yeast host cell comprises both native and heterologous copies of one or more gene encoding the transketolase. The transketolase can be of prokaryotic or eukaryotic origin. In some embodiments, the transketolase is a TKL1 polypeptide which can be encoded by a tkl1 gene. In specific embodiments, the transketolase is derived from Saccharomyces sp., and, in further specific embodiments, is derived from Saccharomyces cerevisiae. In some embodiments, the transketolase can have the amino acid sequence of SEQ ID NO: 152 (or a variant thereof). In additional embodiments, the transketolase is encoded by a heterologous nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 151 or is a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ I D NO: 152 (or a variant thereof).

[0076] In some embodiments, the epimerase has the ability to convert ribulose 5-phosphate into xylulose 5-phosphate, and / or the ability to convert xylulose 5-phosphate into ribulose 5- phosphate. In some embodiments, the epimerase is native or heterologous to the recombinant yeast host cell. In some additional embodiments, the recombinant yeast host cell comprises native and heterologous copies of the genes encoding the epimerase. In some embodiment, the epimerase is of prokaryotic or eukaryotic origin. In some specific embodiment, the epimerase is a RPE1 polypeptide which is encoded by a rpe1 gene. In some further embodiments, the epimerase is derived from Saccharomyces sp., and, in some specific embodiments, is derived from Saccharomyces cerevisiae. In some specific embodiments, the epimerase has the amino acid sequence of SEQ ID NO: 156 (or a variant thereof). In some additional specific embodiments, the epimerase is encoded by a heterologous nucleic acid molecule comprising the nucleic acid of SEQ ID NO: 155 or is a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 156 (or a variant thereof).

[0077] In some embodiments, the isomerase has the ability to convert ribulose 5-phosphate into ribose 5-phosphate, and / or the ability to convert ribose 5-phosphate into ribulose 5- phosphate. In some specific embodiments, the isomerase is native or heterologous to the recombinant yeast host cell. In some additional embodiments, the recombinant yeast host cell comprises native and heterologous copies of the gene encoding the isomerase. In some additional embodiments, the isomerase is of prokaryotic or eukaryotic origin. In some specific embodiments, the isomerase is a RKI1 polypeptide which is encoded by a rki1 gene. In some specific embodiments, the isomerase is derived from Saccharomyces sp., and, in some specific embodiments, is derived from Saccharomyces cerevisiae. In some additional embodiments, the isomerase has the amino acid sequence of SEQ ID NO: 158 (or a variant thereof). In some further additional embodiments, the isomerase is encoded by a heterologous nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 157 or is a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 158 (or a variant thereof).

[0078] In further embodiments, the recombinant yeast host cell comprises at least two copies (per haploid genome) of the transaldolase, the transketolase, the epimerase, and / or the isomerase. In additional embodiments, the recombinant yeast host cell comprises at least three copies (per haploid genome) of the transaldolase, the transketolase, the epimerase, and / or the isomerase. In yet further embodiments, the recombinant yeast host cell comprises the transaldolase, the transketolase, the epimerase, and the isomerase.

[0079] To further increase the production of acetyl-coA in the recombinant yeast host cell, it is possible to increase the conversion of pantothenate into Coenzyme A. In some embodiments, the recombinant yeast host cell comprises native and heterologous copies of the polypeptides involved in the conversion of pantothenate into Coenzyme A. In such embodiment the recombinant yeast host cell of the present disclosure comprises at least one of pantothenate symporter (for example FEN2 which can be encoded by the fen2 gene), a pantothenate kinase (for example CAB1 which can be encoded by the cab1 gene), a phosphopantothenoylcysteine synthetase (for example CAB2 which can be encoded by the cab2 gene), a phosphopantothenoylcysteine decarboxylase (for example CAB3 which can be encoded by the cab3 gene), a pantetheine-phosphate adenylyltransferase (for example CAB4 which can be encoded by the cab4 gene) or a dephospho-CoA kinase (for example CAB5 which can be encoded by the cab5 gene). The FEN2 polypeptide is a plasma membrane proton-pantothenate symporter. The FEN2 polypeptide is classified in 2. A.1.14.18 in the Transporter Classification Database. Increasing the expression of a native and / or a heterologous fen2 gene is expected to increase intracellular entry of pantothenate and, ultimately, favor the conversion of pantothenate into acetyl-coA. The recombinant yeast host cell of the present disclosure can have native and heterologous copies of the fen2 gene encoding the FEN2 polypeptide. In some embodiments, the FEN2 polypeptide is derived from Saccharomyces sp., and, in some additional embodiment, is derived from Saccharomyces cerevisiae. In additional embodiments, the FEN2 polypeptide has the amino acid sequence of SEQ ID NO: 307 (or a variant thereof). In further embodiments, the FEN2 polypeptide is encoded by a heterologous nucleic acid molecule comprising a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 307 (or a variant thereof). As used in the context of the present disclosure, “panthotenate kinases” are enzymes capable of phosphorylating pantothenate into 4’-phosphopantothenate, a precursor of CoenzymeA. In some embodiments, the pantothenate kinase is the CAB1 polypeptide which is encoded by the cab1 gene. In some embodiments, the pantothenate kinase is native or heterologous to the recombinant yeast host cell. In some additional embodiments, the recombinant yeast host cell comprises native and heterologous copies of the gene encoding the pantothenate kinase. In some embodiments, the pantothenate kinase is of prokaryotic or eukaryotic origin. In yet additional embodiment, the pantothenate kinase is derived from Saccharomyces sp., and in some further embodiments, is derived from Saccharomyces cerevisiae. In specific embodiments, the pantothenate kinase has the amino acid sequence of SEQ ID NO: 308(or a variant thereof) or 342 (or a variant thereof). In additional embodiments, the pantothenate kinase is encoded by a heterologous nucleic acid molecule comprising a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 308 (or a variant thereof) or 342 (or a variant thereof). As used in the context of the present disclosure, “phosphopantothenoylcysteine synthetase” are enzymes capable of biosynthesis of coenzyme A (CoA) from pantothenic acid. In some embodiments, the phosphopantothenoylcysteine synthetase is the CAB2 polypeptide which is encoded by the cab2 gene. In some embodiments, the phosphopantothenoylcysteine synthetase is native or heterologous to the recombinant yeast host cell. In some additional embodiments, the recombinant yeast host cell comprises native and heterologous copies of the gene encoding the phosphopantothenoylcysteine synthetase. In some embodiments, the phosphopantothenoylcysteine synthetase is of prokaryotic or eukaryotic origin. In yet additional embodiment, the phosphopantothenoylcysteine synthetase is derived from Saccharomyces sp., and in some further embodiments, is derived from Saccharomyces cerevisiae. In specific embodiments, the phosphopantothenoylcysteine synthetase has the amino acid sequence of SEQ ID NO: 318 (or a variant thereof). In additional embodiments, the phosphopantothenoylcysteine synthetase is encoded by a heterologous nucleic acid molecule comprising a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 318 (or a variant thereof). As used in the context of the present disclosure, “phosphopantothenoylcysteine decarboxylase” are enzymes capable of N-[(R)-4'-phosphopantothenoyl]-L-cysteine into pantotheine 4'- phosphate and carbon dioxide. In some embodiments, the phosphopantothenoylcysteine decarboxylase is the CAB3 polypeptide which is encoded by the cab3 gene. In some embodiments, the phosphopantothenoylcysteine decarboxylase is native or heterologous to the recombinant yeast host cell. In some additional embodiments, the recombinant yeast host cell comprises native and heterologous copies of the gene encoding the phosphopantothenoylcysteine decarboxylase. In some embodiments, the phosphopantothenoylcysteine decarboxylase is of prokaryotic or eukaryotic origin. In yet additional embodiment, the phosphopantothenoylcysteine decarboxylase is derived from Saccharomyces sp., and in some further embodiments, is derived from Saccharomyces cerevisiae. In specific embodiments, the phosphopantothenoylcysteine decarboxylase has the amino acid sequence of SEQ ID NO: 320 (or a variant thereof). In additional embodiments, the phosphopantothenoylcysteine decarboxylase is encoded by a heterologous nucleic acid molecule comprising a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 320 (or a variant thereof). As used in the context of the present disclosure, “pantetheine-phosphate adenylyltransferase” are enzymes that catalyses the magnesium-dependent adenylyl transfer from ATP to 4'- phosphopantetheine (Ppant or PhP) to form dephospho-CoA (dPCoA. In some embodiments, the pantetheine-phosphate adenylyltransferase is the CAB4 polypeptide which is encoded by the cab4 gene. In some embodiments, the pantetheine-phosphate adenylyltransferase is native or heterologous to the recombinant yeast host cell. In some additional embodiments, the recombinant yeast host cell comprises native and heterologous copies of the gene encoding the pantetheine-phosphate adenylyltransferase. In some embodiments, the pantetheine-phosphate adenylyltransferase is of prokaryotic or eukaryotic origin. In yet additional embodiment, the pantetheine-phosphate adenylyltransferase is derived from Saccharomyces sp., and in some further embodiments, is derived from Saccharomyces cerevisiae. In specific embodiments, the pantetheine-phosphate adenylyltransferase has the amino acid sequence of SEQ ID NO: 322 (or a variant thereof). In additional embodiments, the pantetheine-phosphate adenylyltransferase is encoded by a heterologous nucleic acid molecule comprising a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 322 (or a variant thereof). As used in the context of the present disclosure, “dephospho-CoA kinase” are enzymes that convert ATP and dephospho-CoA into ADP and CoA. In some embodiments, the dephospho-CoA kinase is the CAB5 polypeptide which is encoded by the cab5 gene. In some embodiments, the dephospho- CoA kinase is native or heterologous to the recombinant yeast host cell. In some additional embodiments, the recombinant yeast host cell comprises native and heterologous copies of the gene encoding the dephospho-CoA kinase. In some embodiments, the dephospho- CoA kinase is of prokaryotic or eukaryotic origin. In yet additional embodiment, the dephospho-CoA kinase is derived from Saccharomyces sp., and in some further embodiments, is derived from Saccharomyces cerevisiae. In specific embodiments, the dephospho-CoA kinase has the amino acid sequence of SEQ ID NO: 324 (or a variant thereof). In additional embodiments, the dephospho-CoA kinase is encoded by a heterologous nucleic acid molecule comprising a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 324 (or a variant thereof). In yet additional embodiments, the recombinant yeast host cell can comprise the FEN2 polypeptide. In yet further embodiments, the recombinant yeast host cell can comprise the CAB1 , CAB2, CAB3, CAB4, and / or CAB5 polypeptide. In still additional embodiments, the recombinant yeast host cell can comprise the FEN2 and the CAB1 , CAB2, CAB3, CAB4, and / or CAB5 polypeptide.

[0080] In some embodiment, the recombinant yeast host cell of the present disclosure comprises: a. A heterologous acetaldehyde dehydrogenase under the control of one or more heterologous aerobic promoters; and b. Inactivation of a native acetaldehyde dehydrogenase.

[0081] In such embodiment, the recombinant yeast host cell has a lower expression of acetaldehyde dehydrogenase activity during anaerobic conditions when compared to aerobic conditions. In one embodiment, the native and / or heterologous acetaldehyde dehydrogenase is belonging to EC 1.2.1.5. In some embodiments, the native and / or heterologous acetaldehyde dehydrogenase is a mitochondrial acetaldehyde dehydrogenase. In another embodiment, the native and / or heterologous acetaldehyde dehydrogenase is K+-dependent. In some other embodiment, the native and / or heterologous acetaldehyde dehydrogenase can use either NADP+ or NAD+ as cofactor. In some other embodiment, the native and / or heterologous acetaldehyde dehydrogenase is an ALD4. In an embodiment, the heterologous acetaldehyde dehydrogenase is an ALD4 under the control of icl1 aerobic promoter and the inactivated acetaldehyde dehydrogenase is a native ALD4. In some embodiments, the recombinant yeast host cell of the present disclosure has native and / or heterologous acetaldehyde dehydrogenase 4 (ALD4). Acetaldehyde dehydrogenase 4, a specific type of aldehyde dehydrogenase, are classified under Enzyme Commission No. 1.2.1.5 and are intended to encompass enzymes capable of converting acetaldehyde to acetate using NAD+ or NADP+ as cofactors. In other embodiments, the enzyme having acetaldehyde dehydrogenase 4 activity can be encoded by an ald4 gene (e.g., ALD4). In some embodiments, the acetaldehyde dehydrogenase 4 can be native to the recombinant yeast host cell. In other embodiments, the enzyme having acetaldehyde dehydrogenase 4 can be heterologous to the recombinant yeast host cell. In yet other embodiments, the activity associated with the enzyme having acetaldehyde dehydrogenase 4 can be provided from both a native gene and a heterologous one. In an embodiment, the acetaldehyde dehydrogenase 4 can be obtained from Saccharomyces sp., and in further embodiments from Saccharomyces cerevisiae. In such embodiment, the acetaldehyde dehydrogenase 4 can have the amino acid sequence of SEQ ID NO: 346 or be a variant of the amino acid sequence of SEQ ID NO: 346 having acetaldehyde dehydrogenase 4 activity. In addition, the acetaldehyde dehydrogenase 4 can be encoded by the nucleic acid sequence of SEQ I D NO: 345 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 346 (or a variant thereof).

[0082] In some embodiment, the recombinant yeast host cell of the present disclosure can be further engineered to inactivated native CRC1 and / or CIT2 genes.

[0083] [2] In some embodiment, the recombinant yeast host cell of the present disclosure can be further modified by engineering of the pentose phosphate pathway reactions into a cyclic non-oxidative glycolysis cycle by incorporating three native enzymes, the SBPase (SHB17), triosephosphate isomerase (TP11 ), and the fructose-1 ,6-bisphosphate aldolase (FBA1). Multiple variations of the cyclic NOG pathway exist that differ slightly in which enzymatic activities are present.

[0084] [3] In an embodiment, the first engineered metabolic pathway can comprise a step of / means for converting sedoheptulose 1 ,7-bisphosphate to sedoheptulose 7-phosphate. In such embodiment, the first engineered metabolic pathway can comprise a sedoheptulose-bisphosphatase (SBPase). As used herein, the terms "sedoheptulosebisphosphatase" and "SHB17" are intended to include the enzymes capable of converting sedoheptulose 1 ,7-bisphosphate to sedoheptulose 7-phosphate. Sedoheptulose- bisphosphatase include those enzymes that correspond to Enzyme Commission Number 3.1.3.37. In the context of the present disclosure, the enzyme having SBPase activity may be native or heterologous to the recombinant yeast host cell. In some embodiments, the enzyme having SBPase activity is of prokaryotic, fungal or other eukaryotic origin. In other embodiments, the enzyme having SBPase activity can be encoded by a shb17 gene (e.g., SHB17). In an embodiment, the enzyme having SBPase activity is of fungal origin. In an embodiment, the enzyme having SBPase activity is derived from Saccharomyces sp., and in further embodiments from Saccharomyces cerevisiase. In such embodiment, the enzyme having SBPase activity can have the amino acid sequence of SEQ ID NO: 338 or be a variant of the amino acid sequence of SEQ ID NO: 338 having SBPase activity. In addition, the enzyme having SBPase activity can be encoded by the nucleic acid sequence of SEQ ID NO: 337 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 338 (or a variant thereof).

[0085] [4] In an embodiment, the first engineered metabolic pathway can comprise a step of / means for converting D-fructose 1 ,6-bisphosphate into D-glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). In such embodiment, the first engineered metabolic pathway can comprise a fructose 1 ,6-bisphosphate aldolase (FBP aldolase). As used herein, the terms "fructose 1 ,6-bisphosphate aldolase" and "FBP aldolase" are intended to include the enzymes capable of converting fructose 1 ,6- bisphosphate into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. FBP aldolase include those enzymes that correspond to Enzyme Commission Number 4.1.2.13. In the context of the present disclosure, the enzyme having FBP aldolase activity may be native or heterologous to the recombinant yeast host cell. In some embodiments, the enzyme having FBP aldolase activity is of prokaryotic, fungal or other eukaryotic origin. In other embodiments, the enzyme having FBP aldolase activity can be encoded by a fba 1 gene (e.g., FBA1). In an embodiment, the enzyme having FBP aldolase activity is of fungal origin. In an embodiment, the enzyme having FBP aldolase activity is derived from Saccharomyces sp., and in further embodiments from Saccharomyces cerevisiase. In such embodiment, the enzyme having FBP aldolase activity can have the amino acid sequence of SEQ ID NO: 334, or be a variant of the amino acid sequence of SEQ ID NO: 334 having FBP aldolase activity. In addition, the enzyme having FBP aldolase activity can be encoded by the nucleic acid sequence of SEQ ID NO: 333 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 334 (or a variant thereof). In another embodiment, the first engineered metabolic pathway can comprise a step of / means for converting dihydroxyacetone phosphate (DHAP) into D-glyceraldehyde-3- phosphate (G3P). In such embodiment, the first engineered metabolic pathway can comprise a triose phosphate isomerase (TP isomerase). As used herein, the terms "triose phosphate isomerase", “TPI1” and "TP isomerase " are intended to include the enzymes capable of converting dihydroxyacetone phosphate (DHAP) into D-glyceraldehyde-3- phosphate (G3P). TP isomerase include those enzymes that correspond to Enzyme Commission Number 5.3.1 .1 . In the context of the present disclosure, the enzyme having TP isomerase activity may be native or heterologous to the recombinant yeast host cell. In some embodiments, the enzyme having TP isomerase activity is of prokaryotic, fungal or other eukaryotic origin. In other embodiments, the enzyme having TP isomerase activity can be encoded by a tpi1 gene (e.g., TPI1). In an embodiment, the enzyme having TP isomerase activity is of fungal origin. In an embodiment, the enzyme having TP isomerase activity is derived from Saccharomyces sp., and in further embodiments from Saccharomyces cerevisiae. In such embodiment, the enzyme having TP isomerase activity can have the amino acid sequence of SEQ I D NO: 336, or be a variant of the amino acid sequence of SEQ ID NO: 336 having TP isomerase activity. In addition, the enzyme having TP isomerase activity can be encoded by the nucleic acid sequence of SEQ ID NO: 335 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 336 (or a variant thereof).

[0086] In some embodiments, the recombinant yeast host cell of the present disclosure can be used to generate farnesene from acetyl-coA. In such embodiment, the recombinant yeast host cell can include / express at least one of a NADPH or NADH-dependent 3-hydroxy-3- methylglutaryl coenzyme-A (HMG-CoA) reductase (like HMG1 , HMG2, or HMGR for example) or a farnese syntase. In an embodiment, the 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase is native or heterologous to the recombinant yeast host cell. In some specific embodiment, the recombinant yeast host cell comprises native and heterologous copies of the 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase. In an embodiment, the farnesene synthase is native or heterologous to the recombinant yeast host cell. In some specific embodiment, the recombinant yeast host cell comprises native and heterologous copies of the farnesene synthase. Embodiments of recombinant yeast host cells capable of generating farnesene are disclosed in Meadows et al., 2016, which is incorporated herewith in its entirety. In some embodiments, the recombinant yeast host cell of the present disclosure can be used to generate 3-hydroxy-propionic acid from acetyl-coA. In such embodiment, the recombinant yeast host cell can include / express at least one of an acetyl-coA carboxylase (like ACC1 for example) or a malonyl-CoA reductase (MCR). In some embodiments, the recombinant yeast host cell can include / express at least one of a acetyl-coA carboxylase (like ACC1 for example) and a malonyl-CoA reductase (MCR). In some embodiments, the acetyl-coA carboxylase is native or heterologous to the recombinant yeast host cell. In some additional embodiments, the recombinant yeast host cell comprises native and heterologous copies of the gene encoding the acetyl-coA carboxylase. In some embodiments, the malonyl-CoA reductase is native or heterologous to the recombinant yeast host cell. In some additional embodiments, the recombinant yeast host cell comprises native and heterologous copies of the gene encoding the malonyl-CoA reductase. Embodiments of recombinant yeast host cells capable of generating 3-hydroxy- propionic acid are disclosed in Qin et al., 2020, and Hellgren et al., 2020 which are both incorporated herewith in their entirety.

[0087] In some embodiments, the recombinant yeast host cell of the present disclosure can be used to generate polyhydroxybutyrate from acetyl-coA. In some embodiments, the recombinant yeast host cell can include / express at least one of a beta-ketothiolase (PHAA / ERG10), NADPH-dependent acetoacetyl-CoA reductase (PHAB), or polyhydroxyalkanoate synthase (PHAC). In some embodiments, the beta-ketothiolase is native or heterologous to the recombinant yeast host cell. In some additional embodiments, the recombinant yeast host cell comprises native and heterologous copies of the gene encoding the beta-ketothiolase. In some embodiments, the NADPH- dependent acetoacetyl-CoA reductase is native or heterologous to the recombinant yeast host cell. In some additional embodiments, the recombinant yeast host cell comprises native and heterologous copies of the gene encoding the NADPH-dependent acetoacetyl- CoA reductase. In some embodiments, the polyhydroxyalkanoate synthase is native or heterologous to the recombinant yeast host cell. In some additional embodiments, the recombinant yeast host cell comprises native and heterologous copies of the gene encoding the polyhydroxyalkanoate synthase. Embodiments of recombinant yeast host cells capable of generating polyhydroxybutyrate are disclosed in Kocharin et al., 2013, which is incorporated herewith in its entirety. In some embodiments, the recombinant yeast host cell of the present disclosure can be used to generate carotenoid from acetyl-coA. In some embodiments, the recombinant yeast host cell can include / express at least one of a truncated HMG CoA reductase (tHMG1), geranylgeranyl diphosphate synthase (CRTE), phytoene synthase (CRTB), or phytoene desaturase (CRTI). In some embodiments, the truncated HMG CoA reductase is native or heterologous to the recombinant yeast host cell. In some additional embodiments, the recombinant yeast host cell comprises native and heterologous copies of the gene encoding the truncated HMG CoA reductase. In some embodiments, the geranylgeranyl diphosphate synthase is native or heterologous to the recombinant yeast host cell. In some additional embodiments, the recombinant yeast host cell comprises native and heterologous copies of the gene encoding the geranylgeranyl diphosphate synthase. In some embodiments, the phytoene synthase is native or heterologous to the recombinant yeast host cell. In some additional embodiments, the recombinant yeast host cell comprises native and heterologous copies of the gene encoding the phytoene synthase. In some embodiments, the phytoene desaturase is native or heterologous to the recombinant yeast host cell. In some additional embodiments, the recombinant yeast host cell comprises native and heterologous copies of the gene encoding the phytoene desaturase. Embodiments of recombinant yeast host cells capable of generating carotenoids are disclosed in Su et al., 2020, which is incorporated herewith in its entirety.

[0088] In some embodiments, the recombinant yeast host cell of the present disclosure can be used to generate fatty acid ethyl esther from acetyl-coA. In some embodiments, the recombinant yeast host cell can include / express at least one of a wax ester synthase, acetyl-CoA carboxylase (ACC1), fatty acid synthase (FAS1 and / or FAS2). In some embodiments, the wax ester synthase is native or heterologous to the recombinant yeast host cell. In some additional embodiments, the recombinant yeast host cell comprises native and heterologous copies of the gene encoding the wax ester synthase. In some embodiments, the acetyl-CoA carboxylase is native or heterologous to the recombinant yeast host cell. In some additional embodiments, the recombinant yeast host cell comprises native and heterologous copies of the gene encoding the acetyl-CoA carboxylase. In some embodiments, the fatty acid synthase is native or heterologous to the recombinant yeast host cell. In some additional embodiments, the recombinant yeast host cell comprises native and heterologous copies of the gene encoding the fatty acid synthase. Embodiments of recombinant yeast host cells capable of generating fatty acid esters are disclosed in de Jong et al., 2014, which is incorporated herewith in its entirety. In some embodiments, the recombinant yeast host cell of the present disclosure can be used to generate ethanol from acetyl-coA. Embodiments of recombinant yeast host cells capable of generating ethanol and including further genetic modifications are disclosed in US20130273555, US20130273555, US20130273555, US20160194669, US8871488, US9790521 , US9181566, US9422581 , US9422582, US9550999, US10240168,

[0089] US10006058, US20170275652, US10428354, US20190338256, US20200377559, US20210047660, US11447783, US20210147792, US20210207076, US20210221857, US20210292734, US20210395756, US20210388397, US20210332091 ,

[0090] US20220251608, US20220251582, US20220259604, US20230002793,

[0091] US20230331789, US20210380989, PCT / IB2022 / 059754, US11753656,

[0092] US20200224209, US11332728, US11198881 , US10570421 , US20230193232, PCT / IB2023 / 052263, and US20230091532 which are all incorporated in their entirety.

[0093] Process of using the recombinant yeast host cell

[0094] The present disclosure provides a process for converting a carbohydrate into a fermentation product by a recombinant yeast host cell comprising at least (a) an upregulated pyruvate kinase, (b) a first engineered metabolic pathway to convert the carbohydrate into acetyl-coA comprising a heterologous polypeptide having phosphoketolase activity, and (c) a second engineered metabolic pathway to convert acetyl-CoA into the fermentation product. The fermentation kinetic observed when culturing the recombinant yeast host cell is increased when compared to the fermentation kinetic of a control yeast host cell comprising the first and the second engineered metabolic pathways as defined in (b) and (c) but lacking the upregulated pyruvate kinase as defined in (a).

[0095] Broadly, the process of the present disclosure comprises contacting the recombinant yeast host cell with a carbohydrate under conditions to allow the conversion of at least in part of the carbohydrate into fermentation product (e.g., fermenting step). The carbohydrate is present in a fermentation medium (sometimes referred to as a biomass). The process can optionally include a step of isolating the one or more fermentation product from the fermented fermentation medium (using distillation for example). The process can also optionally include a step of recuperating the distiller’s grain for animal nutrition. In some embodiments, the process can include, prior to the contacting step, a step of propagating the recombinant yeast host cell. The one or more fermentation product can comprise, without limitation, acetone, farnesene, 3-hydroxy-propionic acid, p-coumaric acid, 2-phenylethanol, tryosol, salidroside, polyhydroxybutyrate, carotenoid, a fatty acid ethyl esther, isopropanol, ethanol or combinations thereof. In some embodiments, the fermentation products can comprise at least two distinct fermentation products such as at least two of any one of the following: acetone, farnesene, 3-hydroxy-propionic acid, p-coumaric acid, 2-phenylethanol, tryosol, salidroside, polyhydroxybutyrate, carotenoid, a fatty acid ethyl esther, isopropanol, or ethanol. In some specific embodiments, the fermentation product can comprise acetone, isopropanol, and / or ethanol. In some embodiments, the fermentation products can comprise at least three distinct fermentation products such as at least three of any one of the following: acetone, farnesene, 3-hydroxy-propionic acid, p-coumaric acid, 2- phenylethanol, tryosol, salidroside, polyhydroxybutyrate, carotenoid, a fatty acid ethyl esther, isopropanol, or ethanol. In some specific embodiments, the fermentation product can comprise acetone and ethanol. In another embodiments, the fermentation product comprises ethanol.

[0096] In some embodiments, the process of the present disclosure comprises a plurality of fermentations in which the recombinant yeast host cells are recycled between two rounds of fermentations. In some embodiments, the recombinant yeast host cells are only exogenously added in the initial fermentation cycle and are then recycled in further fermentation cycles. Each fermentation cycle of the process includes contacting a fermentation medium (comprising a fermentable carbohydrate) with a fermenting population under conditions so as to allow the conversion of the fermentable carbohydrate in a fermentation product (e.g., fermentation). At the end of the fermentation, the fermenting population present in the fermented fermentation medium is substantially isolated from the fermented fermentation medium and use to initiate another fermentation cycle. It is understood that, in such embodiments, the initial fermenting population consists essentially in the recombinant yeast host cells of the present disclosure and that, during the plurality of the fermentation cycles, the recycled fermenting population can include some contaminating wild (non-genetically modified) yeasts. The plurality of fermentation cycles can include at least one continuous fermentation. The plurality of fermentation cycles can only include continuous fermentations. The plurality of fermentation cycles can include at least one batch fermentation. The plurality of fermentation cycles can only include batch fermentations. The processes of the present disclosure can include an initial fermentation cycle at least one, two, three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 39 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 49 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 59 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 69 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 79 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 89 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 99 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 109 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 119 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 129 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 139 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 149 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 159 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 169 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 179 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 189 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 199 further fermentation cycles.

[0097] Each fermentation cycle (irrespective of its type) comprises a step of contacting the fermentation medium with a fermenting population of recombinant yeast host cells. The fermenting population will ferment (e.g., convert some of the biomass into a fermentation product) the fermentation medium to generate a fermented medium. The contacting step can include simultaneously adding the fermenting population and the fermentation medium to a fermenter. The contacting step can include adding the fermentation medium to a fermenter and subsequently adding the fermenting population to the fermentation medium. The contacting step can include adding fermentation medium to a fermenter already containing the fermentation population. After the fermentation, a fermented medium will be obtained, and it comprises a fermentation product and a fermented population. In a process comprising a batch fermentation cycle, the fermenting population is contacted (e.g., pitched) with a fermentation medium in a fermenter. The fermenting population can be added to the fermenter prior to, at the same time and / or after the fermentation medium has been added. In a process comprising a continuous fermentation cycle, the fermenting population can be added to the series of fermenters prior to, at the same time and / or after the fermentation medium has been added.

[0098] The fermentation medium that can be fermented by the recombinant yeast host cells described herein includes any type of fermentable carbohydrate known in the art and described herein (e.g. glucose). In some embodiment, the carbohydrate is a complex carbohydrate that could be saccharify in simple sugar such as glucose (e.g. cellulose, sucrose, trehalose, xylose, and / or arabinose). In one embodiment, the carbohydrate source is a biomass. For example, the biomass can include, but is not limited to, starch, sugar and lignocellulosic materials comprising lignocellulosic fibers. Starch materials can include, but are not limited to, mashes such as corn, wheat, rye, barley, rice, or milo. The starch present in the biomass can be totally or in part in a raw form or in a gelatinized form. When the biomass comprises or is derived from corn, it can include a corn mash. Sugar materials can include, but are not limited to, sugar beets, artichoke tubers, sweet sorghum, molasses or cane. The terms “lignocellulosic material”, “lignocellulosic substrate” and “cellulosic biomass” mean any type of biomass comprising cellulose, hemicellulose, lignin, or combinations thereof, such as but not limited to woody biomass, forage grasses, herbaceous energy crops, non-woody-plant biomass, agricultural wastes and / or agricultural residues, forestry residues and / or forestry wastes, paper-production sludge and / or waste paper sludge, waste -water-treatment sludge, municipal solid waste, corn fiber from wet and dry mill corn ethanol plants and sugar-processing residues. The terms “hemicellulosics”, “hemicellulosic portions” and “hemicellulosic fractions” mean the non-lignin, non-cellulose elements of lignocellulosic material, such as but not limited to hemicellulose (i.e., comprising mannan, glucomannan and galactoglucomannan), pectins (e.g., homogalacturonans, rhamnogalacturonan I and II, and xylogalacturonan) and proteoglycans (e.g., arabinogalactan-protein). In some embodiments, the biomass can include and / or be supplemented with citric acid (especially when acetic acid or acetate is the first metabolic product).

[0099] In a non-limiting example, the lignocellulosic material can include, but is not limited to, woody biomass, such as recycled wood pulp fiber, sawdust, hardwood, softwood, and combinations thereof; grasses, such as switch grass, cord grass, rye grass, reed canary grass, miscanthus, or a combination thereof; sugar-processing residues, such as but not limited to sugar cane bagasse; sugar cane must; agricultural wastes, such as but not limited to rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, and corn fiber; stover, such as but not limited to soybean stover, corn stover; succulents, such as but not limited to, agave; and forestry wastes, such as but not limited to, recycled wood pulp fiber, sawdust, hardwood (e.g., poplar, oak, maple, birch, willow), softwood, or any combination thereof. Lignocellulosic material may comprise one species of fiber; alternatively, lignocellulosic material may comprise a mixture of fibers that originate from different lignocellulosic materials. Other lignocellulosic materials are agricultural wastes, such as cereal straws, including wheat straw, barley straw, canola straw and oat straw; corn fiber; stovers, such as corn stover and soybean stover; grasses, such as switch grass, reed canary grass, cord grass, and miscanthus; or combinations thereof.

[0100] Substrates for cellulose activity assays can be divided into two categories, soluble and insoluble, based on their solubility in water. Soluble substrates include cellodextrins or derivatives, carboxymethyl cellulose (CMC), or hydroxyethyl cellulose (HEC). Insoluble substrates include crystalline cellulose, microcrystalline cellulose (Avicel), amorphous cellulose, such as phosphoric acid swollen cellulose (PASC), dyed or fluorescent cellulose, and pretreated lignocellulosic biomass. These substrates are generally highly ordered cellulosic material and thus only sparingly soluble.

[0101] It will be appreciated that suitable lignocellulosic material may be any feedstock that contains soluble and / or insoluble cellulose, where the insoluble cellulose may be in a crystalline or non-crystalline form. In various embodiments, the lignocellulosic biomass comprises, for example, wood, corn, corn stover, sawdust, bark, molasses, sugarcane, leaves, agricultural and forestry residues, grasses such as switchgrass, ruminant digestion products, municipal wastes, paper mill effluent, newspaper, cardboard or combinations thereof. Paper sludge is also a viable feedstock for lactate or acetate production. Paper sludge is solid residue arising from pulping and paper-making and is typically removed from process wastewater in a primary clarifier. The cost of disposing of wet sludge is a significant incentive to convert the material for other uses, such as conversion to ethanol. Processes provided by the present invention are widely applicable. Moreover, the saccharification and / or fermentation products may be used to produce ethanol and acetone or higher value-added chemicals, such as organic acids, aromatics, esters, and polymer intermediates.

[0102] In specific embodiments, the fermentation medium comprises sugarcane or a sugarcane derivative. After it has been harvested, the sugarcane is pressed or diffused to generate sugarcane juice and a solid fibrous residue, the cane bagasse. In the context of the present disclosure, sugarcane juice is considered to be a sugarcane derivative. The sugarcane juice can be clarified and concentrated by evaporation until sucrose crystallization is observed. The clarified sugarcane juice and the concentrated sugarcane juice are considered sugarcane derivatives. The sucrose crystals obtained after crystallization can be collected by centrifugation, generating a sucrose saturated viscous phase, called “cane molasses”. Cane molasses, which is also considered to be a sugarcane derivative, can include between 45 to 60 % sucrose and 5 to 20 % glucose plus fructose. In some embodiments, the fermentation medium comprises, as a sugarcane derivative, a sugarcane juice. In another embodiment, the fermentation medium comprises, as a sugarcane derivative, a cane molasses. In still another embodiment, the fermentation medium comprises, as a sugarcane derivative, both sugarcane juice and a cane molasses.

[0103] The fermentation step of the process can be performed at temperatures of at least about 25°C, about 28°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41 °C, about 42°C, or about 50°C. In some embodiments, the process can be conducted at temperatures above about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41 °C, about 42°C, or about 50°C.

[0104] In some embodiments, prior to fermentation, a step of liquefying starch can be included in the process. In such embodiment, the liquefied starch is then submitted to a following fermentation step. The liquefaction of starch can be performed at a temperature of between about 70°C-105°C to allow for proper gelatinization and hydrolysis of the starch. In an embodiment, the liquefaction occurs at a temperature of at least about 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C or 105°C. Alternatively, or in combination, the liquefaction occurs at a temperate of no more than about 105°C, 100°C, 95°C, 90°C, 85°C, 80°C, 75°C or 70°C. In yet another embodiment, the liquefaction occurs at a temperature between about 80°C and 85°C (which can include a thermal treatment spike at 105°C).

[0105] During fermentation, the pH of the fermentation medium can be equal to or below 5.5, 5.4, 5.3, 5.2, 5.1 , 5.0, 4.9, 4.8, 4.7., 4.6, 4.5, 4.4, 4.3, 4.2, 4.1 , 4.0 or lower. In an embodiment, the pH of the fermentation medium (during fermentation) is between 4.0 and 5.5.

[0106] In some embodiments, the processes of the present disclosure comprise producing the fermentation product at a particular rate. For example, in some embodiments, the fermentation product is produced at a rate of at least about 0.1 mg per hour per liter, at least about 0.25 mg per hour per liter, at least about 0.5 mg per hour per liter, at least about 0.75 mg per hour per liter, at least about 1.0 mg per hour per liter, at least about 2.0 mg per hour per liter, at least about 5.0 mg per hour per liter, at least about 10 mg per hour per liter, at least about 15 mg per hour per liter, at least about 20.0 mg per hour per liter, at least about 25 mg per hour per liter, at least about 30 mg per hour per liter, at least about 50 mg per hour per liter, at least about 100 mg per hour per liter, at least about 200 mg per hour per liter, at least about 300 mg per hour per liter, at least about 400 mg per hour per liter, at least about 500 mg per hour per liter, at least about 600 mg per hour per liter, at least about 700 mg per hour per liter, at least about 800 mg per hour per liter, at least about 900 mg per hour per liter, at least about 1 g per hour per liter, at least about 1.5 g per hour per liter, at least about 2 g per hour per liter, at least about 2.5 g per hour per liter, at least about 3 g per hour per liter, at least about 3.5 g per hour per liter, at least about 4 g per hour per liter, at least about 4.5 g per hour per liter, at least about 5 g per hour per liter, at least about 5.5 g per hour per liter, at least about 6 g per hour per liter, at least about 6.5 g per hour per liter, at least about 7 g per hour per liter, at least about 7.5 g per hour per liter, at least about 8 g per hour per liter, at least about 8.5 g per hour per liter, at least about 9 g per hour per liter, at least about 9.5 g per hour per liter, at least about 10 g per hour per liter, at least about 10.5 g per hour per liter, at least about 11 g per hour per liter, at least about 11.5 g per hour per liter, at least about 12 g per hour per liter, at least about 12.5 g per hour per liter, at least about 13 g per hour per liter, at least about 13.5 g per hour per liter, at least about 14 g per hour per liter, at least about 14.5 g per hour per liter or at least about 15 g per hour per liter.

[0107] In some embodiments, the processes of the present disclosure can produce the fermentation product at a rate of at least about 0.1 mg per hour per liter, at least about 0.25 mg per hour per liter, at least about 0.5 mg per hour per liter, at least about 0.75 mg per hour per liter, at least about 1.0 mg per hour per liter, at least about 2.0 mg per hour per liter, at least about 5.0 mg per hour per liter, at least about 10 mg per hour per liter, at least about 15 mg per hour per liter, at least about 20.0 mg per hour per liter, at least about 25 mg per hour per liter, at least about 30 mg per hour per liter, at least about 50 mg per hour per liter, at least about 100 mg per hour per liter, at least about 200 mg per hour per liter, at least about 300 mg per hour per liter, at least about 400 mg per hour per liter, at least about 500 mg per hour per liter, at least about 600 mg per hour per liter, at least about 700 mg per hour per liter, at least about 800 mg per hour per liter, at least about 900 mg per hour per liter, at least about 1 g per hour per liter, at least about 1.5 g per hour per liter, at least about 2 g per hour per liter, at least about 2.5 g per hour per liter, at least about 3 g per hour per liter, at least about 3.5 g per hour per liter, at least about 4 g per hour per liter, at least about 4.5 g per hour per liter, at least about 5 g per hour per liter, at least about 5.5 g per hour per liter, at least about 6 g per hour per liter, at least about 6.5 g per hour per liter, at least about 7 g per hour per liter, at least about 7.5 g per hour per liter, at least about 8 g per hour per liter, at least about 8.5 g per hour per liter, at least about 9 g per hour per liter, at least about 9.5 g per hour per liter, at least about 10 g per hour per liter, at least about 10.5 g per hour per liter, at least about 11 g per hour per liter, at least about 11.5 g per hour per liter, at least about 12 g per hour per liter, at least about 12.5 g per hour per liter, at least about 13 g per hour per liter, at least about 13.5 g per hour per liter, at least about 14 g per hour per liter, at least about 14.5 g per hour per liter, at least about 15 g per hour per liter or more than a control strain (e.g., a wild-type strain or a parental strain) and grown under the same conditions.

[0108] The production of the fermentation product can be measured using any method known in the art. For example, the quantity of ethanol and / or acetone in fermentation samples can be assessed using HPLC analysis. Many ethanol assay kits are commercially available that use, for example, alcohol oxidase enzyme-based assays.

[0109] In the process described herein, it is possible to add an exogenous source (e.g., to dose) of an enzyme to facilitate saccharification or improve fermentation yield. As such, the process can comprise including one or more dose of one or more exogenous enzyme during the liquefaction / saccharification and / or the fermentation step. The exogenous enzyme can be provided in a purified form or in combination with other enzymes (e.g., a cocktail). In the context of the present disclosure, the term “exogenous” refers to a characteristic of the enzyme, namely that it has not been produced during the saccharification or the fermentation step, but that it was produced prior to the saccharification or the fermentation step. The exogenous enzyme that can be used during the saccharification / fermentation process can include, without limitation, an alphaamylase, a glucoamylase, a protease, a phytase, a pullulanase, a cellulase, a xylanase, a trehalase, or any combination thereof.

[0110] In the process described herein, it is possible to add a nitrogen source (usually urea or ammonia) to facilitate liquefaction / saccharification or improve fermentation yield. As such, the process can comprise including one or more amount of the nitrogen source prior to or during the saccharification and / or the fermentation step.

[0111] The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

[0112] EXAMPLE

[0113] EXAMPLE

[0114] Example I

[0115] The genotypes of the various Saccharomyces cerevisiae strains characterized in the Example I are presented in Table 1.

[0116] Table 1. Genetic modifications of the strains characterized in Example I. All the strains were derived from a non-genetically modified Saccharomyces cerevisiae strain (e.g., SC- 1).

[0117] Each strain was propagated overnight in YPD medium, OD normalized, and inoculated at 0.12 OD600 / kg into 30g of corn mash at 31.09% total solids (TS). Mash fermentations were supplemented with 474 ppm Urea as a nitrogen source and 45 AGU / g TS of glucoamylase was added to begin the fermentation. CO2produced during fermentation was monitored by an automated CO2analysis (ACAN) system, Figure 2. The ACAN consists of a custom platform for 60mL serum bottles with tubes connected to a needle in the septum of the serum bottle, which is then connected to 0 to 10mL / min mass flow meter via multichannel HPLC valves and solenoids to allow for multiplexing the measurement of multiple vessels. A computer program controls the valves and solenoids while recording data for each bottle individually, and continually cycles between all active bottles. At the end of the fermentation the collected data is graphed by CO2production rates (mL / min) and the integral of those rates to estimate the total CO2produced during the fermentation (mL). Metabolite production was analyzed via an end point HPLC collected at 72 hours,

[0118] Figure 3.

[0119] Overexpression of either the wild-type SC-3 or SC-4 variant resulted in significantly improved fermentation kinetics compared to strain SC-2 lacking upregulated PYK. In addition, the result also demonstrates that PYK upregulation reduced residual glucose levels at the end of fermentation, resulting in slight increases to ethanol and acetone titers while maintaining glycerol levels of the parental strain.

[0120] Example 2

[0121] The genotypes of the various Saccharomyces cerevisiae strains characterized in the Example 2 are presented in Table 2. Table 2. Genetic modifications of the strains characterized in Example II. All the strains were derived from a non-genetically modified Saccharomyces cerevisiae strain (e.g., SC- 1).

[0122] Each strain was propagated overnight in YPD medium, OD normalized, and inoculated at 0.12 OD600 / kg into 30g of corn mash, in a sealed 60ml serum bottle, at 31.98% total solids (TS). Mash fermentations were supplemented with 474 ppm Urea as a nitrogen source and 45 AGU / g TS of glucoamylase was added to begin the fermentation. Fermentation was carried out at 33°C for the first 24 hours and then reduced to 31 °C for the remainder of fermentation with constant shaking at 175 RPM. CO2produced during fermentation was monitored by an automated CO2analysis (ACAN) system as described above, Figure 4. Metabolite production was analyzed via an end point HPLC collected at 72 hours, Figure 5.

[0123] REFERENCES

[0124] Xu, Y.-F., Zhao, X., Glass, D.S., Absalan, F., Perlman, D.H., Broach, J.R., Rabinowitz, J.D., 2012. Regulation of yeast pyruvate kinase by ultrasensitive allostery independent of phosphorylation. Mol. Cell 48, 52-62.

[0125] Chandel, N.S., 2021. Glycolysis. Cold Spring Harb. Perspect. Biol. 13:a040535

[0126] Meadows, A. L., Hawkins, K.M., Tsegaye, Y, Antipov, E., Kim, Y, Raetz, L., Dahl, R.H., Tai, A., Mahatdejkul-Meadows, T., Xu, L. and Zhao, L., 2016. Rewriting yeast central carbon metabolism for industrial isoprenoid production. Nature, 537(7622), pp.694-697.

[0127] Qin, N., Li, L., Ji, X., Li, X., Zhang, Y, Larsson, C., Chen, Y, Nielsen, J. and Liu, Z., 2020. Rewiring central carbon metabolism ensures increased provision of acetyl-CoA and NADPH required for 3-OH-propionic acid production. ACS Synthetic Biology, 9(12), pp.3236-3244.

[0128] De Jong, B.W., Shi, S., Siewers, V. and Nielsen, J., 2014. Improved production of fatty acid ethyl esters in Saccharomyces cerevisiae through up-regulation of the ethanol degradation pathway and expression of the heterologous phosphoketolase pathway. Microbial cell factories, 13(1), pp.1-10. Kocharin, K., Siewers, V. and Nielsen, J., 2013. Improved polyhydroxybutyrate production by Saccharomyces cerevisiae through the use of the phosphoketolase pathway. Biotechnology and bioengineering, 110(8), pp.2216-2224.

[0129] Su, B., Song, D. and Zhu, H., 2020. Metabolic engineering of Saccharomyces cerevisiae for enhanced carotenoid production from xylose-glucose mixtures. Frontiers in Bioengineering and Biotechnology, 8, p.435.

[0130] Hellgren, J., Godina, A., Nielsen, J. and Siewers, V., 2020. Promiscuous phosphoketolase and metabolic rewiring enables novel non-oxidative glycolysis in yeast for high-yield production of acetyl-CoA derived products. Metabolic engineering, 62, pp.150-160.

Claims

WHAT IS CLAIMED IS:

1. A recombinant yeast host cell for converting a carbohydrate into a fermentation product comprising: a) an upregulated pyruvate kinase; b) a first engineered metabolic pathway to convert the carbohydrate into acetyl-coA comprising a heterologous polypeptide having phosphoketolase activity; and c) a second engineered metabolic pathway to convert acetyl-CoA into the fermentation product.

2. The recombinant yeast host cell of claim 1 , wherein the upregulated pyruvate kinase is a heterologous pyruvate kinase.

3. The recombinant yeast host cell of claim 2, wherein the heterologous pyruvate kinase is derived from a Saccharomyces cerevisiae pyruvate kinase.

4. The recombinant yeast host cell of either claims 2 or 3, wherein the heterologous pyruvate kinase is a pyruvate kinase 1 (PYK1) or a pyruvate kinase 2 (PYK2).

5. The recombinant yeast host cell of any one of claims 2 to 4, wherein the heterologous pyruvate kinase has the amino acid sequence of SEQ ID NO: 312, 314 or 316, or is a variant of the SEQ ID NO: 312, 314 or 316 having a pyruvate kinase activity.

6. The recombinant yeast host cell any one of claims 1 to 5, wherein the heterologous polypeptide having phosphoketolase activity:• is a phosphoketolase classified under Enzyme Commission No. 4.1.2.9 or 4.1.2.22;• has the ability to convert D-xylulose 5-phosphate into D- glyceraldehyde 3-phosphate and acetyl-phosphate;• has the ability to convert D-fructose 6-phosphate into D-erythrose 4-phosphate;• has the ability to convert D-sedoheptulose 7-phosphate into D- ribose 5-phosphate;• has single- or multiple-specificity;• is of prokaryotic or eukaryotic origin;• is encoded by a phk1 gene or a phk2 gene;• is derived from Bifidobacterium, Lactiplantibacillus, Leuconostoc, Penicillium, Aspergillus, Oenococcus, Clostridium or Neurospora species;• is derived from Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium gallicum, Bifidobacterium animalis, Bifidobacterium adolescentis, Bifidobacterium asteroides, Bifidobacterium longum, Lactobacillus pentosus, Lactobacillus acidophilus, Lactobacillus casei, Lactiplantibacillus plantarum, Penicillium chrysogenum, Aspergillus niger, Aspergillus nidulans, Aspergillus clavatus, Neurospora crassa, Leuconostoc mesenteroides, Clostridium acetobutylicum or Oenococcus oeni• has the amino acid sequence of SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 or 70, or is a variant of the SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, or 344 having a phosphoketolase activity; and / or• is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, or 343 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, or 344.

7. The recombinant yeast host cell any one of claims 1 to 6, wherein the heterologous phosphoketolase has at least the ability to convert D-fructose 6-phosphate into D- erythrose 4-phosphate.

8. The recombinant yeast host of any one of claims 1 to 7, wherein the fermentation product comprises acetone, isopropanol, and / or ethanol.

9. The recombinant yeast host of claim 8, wherein the fermentation product comprises acetone and / or isopropanol.

10. The recombinant yeast host of claims 8 or 9, wherein the second engineered pathway comprises: i. a thiolase; ii. a CoA transferase, a HMG-CoA synthase and lyase, or an acetoacetyl-CoA hydrolase;iii. an acetoacetate decarboxylase; and iv. optionally a first alcohol dehydrogenase.11 . The recombinant yeast host cell of claiml 0, wherein the thiolase is:• heterologous;• of prokaryotic or eukaryotic origin;• encoded by a th! gene, an erg10 gene or a phaA gene;• derived from Clostridium, Saccharomyces, Cupriavidus, Clostridium,Yarrowia, Thermoanaerobacterium, Saccoglossus,Strongylocentrotus, Zygosaccharomyces or Paenibacillus species;• derived from Clostridium acetobutylicum, Clostridium beijerinckii, Saccharomyces cerevisiae, Cupriavidus necator, Clostridium kluyveri, Yarrowia lipolytica, Thermoanaerobacterium thermosaccharolyticum, Saccoglossus kowalevskii, Strongylocentrotus purpuratus, Zygosaccharomyces bailii or Paenibacillus polymyxa and / or• has the amino acid sequence of SEQ ID NO: 144, 162, 164, 166, 168, 170, 172, 174, 176, 178 or 180, or is a variant of the SEQ ID NO: 144, 162, 164, 166, 168, 170, 172, 174, 176, 178 or 180 having a thiolase activity; and / or• is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 143, 161 , 163, 165, 167, 169, 171 , 173, 175, 177 or 179, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 144, 162, 164, 166, 168, 170, 172, 174, 176, 178 or 180.

12. The recombinant microorganism of claims 10 or 11 , wherein the CoA transferase is:• heterologous;• of prokaryotic or eukaryotic origin;• encoded by a ctfA gene, a ctfB gene, an atoD and / or an atoA gene;• derived from Clostridium, Thermosipho, Escherichia, Paenibacillus, Alkaliphilus or Brevibacillus species;• derived from Clostridium acetobutylicum, Thermosipho melanesiensis, Escherichia coli, Paenibacillus polymyxa, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum, Clostridium sticklandii,Alkaliphilus metalliredigens, or Clostridium bovifaecis or Brevibacillus laterosporus’,• has the amino acid sequence of SEQ ID NO: 146, 148, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214 or 216, or is a variant of the SEQ ID NO: 146, 148, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214 or 216, having a CoA transferase activity; and / or• is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 145, 147, 185, 187, 189, 191 , 193, 195, 197, 199, 201 , 203, 205, 207, 209, 211 , 213 or 215, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 146, 148, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214 or 216.

13. The recombinant microorganism of any one of claims 10 to 12, wherein the HMG- CoA synthase HMG-CoA synthase is:• heterologous;• of prokaryotic or eukaryotic origin;• encoded by a hgms gene or an erg13 gene;• derived from Saccharomyces, Lacticaseibacillus, Enterococcus, Haloferax, Alloscardovia or Listeria’,• derived from Saccharomyces cerevisiae, Lacticaseibacillus casei, Enterococcus faecalis, Haloferax volcanii, Alloscardovia theropitheci, or Listeria monocytogenes-,• has the amino acid sequence of SEQ ID NO: 240, 242, 244, 246, 248 or 250, or is a variant of the SEQ ID NO: 240, 242, 244, 246, 248 or 250 having a HMG-CoA synthase activity; and / or• is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 239, 241 , 243, 245, 247 or 249, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 240, 242, 244, 246, 248 or 250.

14. The recombinant microorganism of any one of claims 10 to 13, wherein the HMG- CoA lyase is:• heterologous;• of prokaryotic or eukaryotic origin;• encoded by an hmgcl gene;• derived from Pseudomonas, Azotobacter, Bacillus, Desulfotomaculum, Acinetobacter, Moraxella, Alcaligenaceae, Macaca, Arabidopsis, Gallus, or Danio species’,• derived from Pseudomonas monteilii, Pseudomonas wayambapalatensis, Azotobacter vinelandii, Pseudomonas citronellolis, Pseudomonas cremoris, Pseudomonas chengduensis, Pseudomonas aeruginosa, Bacillus subtilis, Desulfotomaculum arcticum, Desulfoscipio geothermicus, Acinetobacter baumannii, Acinetobacter Iwoffii, Moraxella caviae, Alcaligenaceae bacterium, Macaca fascicularis, Arabidopsis thaliana, Gallus gall us, or Danio rerio\• has the amino acid sequence of SEQ ID NO: 252, 254, 256, 258, 260,262, 263, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, or 286, or is a variant of the SEQ ID NO: 252, 254, 256, 258, 260, 262,263, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, or 286having a HMG-CoA lyase activity; and / or• is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 251 , 253, 255, 257, 259, 261 , 263, 265, 267, 269, 271 , 273, 275, 277, 279, 281 , 283 or 285, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 252, 254, 256, 258, 260, 262, 263, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, or 286.

15. The recombinant microorganism of any one of claims 10 to 14, wherein the acetoacetyl-CoA hydrolase is:• heterologous;• of prokaryotic or eukaryotic origin;• encoded by a tesB gene, a yciA gene, a yigl gene, a ydil gene, a fadM1 gene, a fadM2 gene ora fadM gene;• derived from Escherichia, Methylorubrum, Pseudomonas, Campylobacter, Mycobacterium, Fibrobacter, Alcanivorax, Haemophilus, Zymomonas, Providencia or Prevotella• derived from Escherichia coll, Methylorubrum extorquens, Pseudomonas aeruginosa, Campylobacter jejuni, Mycobacterium tuberculosis, Pseudomonas putida, Fibrobacter succinogenes,Alcanivorax borkumensis, Haemophilus influenzae, Zymomonas mobilis subsp. Mobilis ZM4, Campylobacter jejuni, Prevotella ruminicola or Providencia sneebia and / or• has the amino acid sequence of SEQ ID NO: 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302 or 303, or is a variant of the SEQ ID NO: 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302 or 303 having an acetoacetyl-CoA hydrolase activity; and / or• is encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302 or 303.

16. The recombinant microorganism of any one of claims 10 to 15, wherein the acetoacetate decarboxylase is:• heterologous;• of prokaryotic or eukaryotic origin;• encoded by an adc gene;• derived from Clostridium, Bacillus, Lacticaseibacillus, Rhizobium, Bradyrhizobium, Tetrahymena, Aspergillus or Paenibacillus species;• derived from Clostridium acetobutylicum, Clostridium beijerinckii,Bacillus amyloliquefaciensi Lacticaseibacillus casei, Lacticaseibacillus rhamnosus, Rhizobium leguminosarum, Bradyrhizobium japonicum, Tetrahymena thermophile, Aspergillus bertholletiae, Aspergillus niger or Paenibacillus polymyxa• has the amino acid sequence of SEQ ID NO: 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, or 238, or is a variant of the SEQ ID NO: 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, or 238 having an acetoacetate decarboxylase activity; and / or• is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 217, 219, 221 , 223, 225, 227, 229, 231 , 233, 235 or 237, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, or 238.

17. The recombinant microorganism of any one of claims 10 to 16, wherein the first alcohol dehydrogenase is:• native and / or heterologous;• of prokaryotic or eukaryotic origin;• encoded by an adh gene;• derived from Zymomonas or Saccharomyces species;• derived from Saccharomyces cerevisiae or Zymomonas mobilise• has the amino acid sequence of SEQ ID NO: 304, 305, or 306 or is a variant of the SEQ ID NO: 304, 305, or 306 having an alcohol dehydrogenase activity; and / or• is encoded by a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 304, 305, or 306.

18. The recombinant yeast host cell of any one of claims 1 to 17, wherein the first engineered metabolic pathway further comprises a heterologous phosphotransacetylase, a heterologous acetate kinase, and / or a heterologous acetyl CoA synthetase.

19. The recombinant yeast host cell of claim 18, wherein the heterologous phosphotransacetylase is:• of prokaryotic or eukaryotic origin;• encoded by a pta gene;• derived from Bifidobacterium, Leuconostoc, Oenococcus, Azotobacter, Lacticaseibacillus, Bacillus, Salmonella, Clostridium, Phytophthora, Globisporangium or Holophagae species;• derived from Bifidobacterium adolescentis, Bifidobacterium animalis, Leuconostoc mesenteroides, Oenococcus oenii, Azotobacter vinelandii, Lactiplantibacillus plantarum, Bacillus subtilis, Salmonella enterica, Clostridium kluyveri, Clostridium phytofermentans, Phytophthora ramorum, Globisporangium splendens or Holophagae bacterium’,• has the amino acid sequence of SEQ ID NO: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 or 102, or is a variant of the SEQ ID NO: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 or 102 having a phosphotransacetylase activity; and / or• is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 75, 77, 79, 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99, or 101 , or adegenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 or 102.

20. The recombinant yeast host cell of either claims 18 or 19, wherein the heterologous acetate kinase is:• of prokaryotic or eukaryotic origin;• encoded by an ack gene;• derived from Bifidobacterium, Leuconostoc, Oenococcus, Escherichia, Bacillus, Clostridium, Salmonella, Phytophthora, Chlamydomonas, Aspergillus or Lactiplantibacillus, species;• derived from Bifidobacterium adolescentis, Leuconostoc mesenteroides, Oenococcus oenii, Escherichia coll, Bacillus subtilis, Clostridium acetobutylicum, Salmonella enterica, Phytophthora ramorum, Chlamydomonas reinhardtii, Aspergillus nidulans, Lactiplantibacillus plantarum or Clostridium kluveryr,• has the amino acid sequence of SEQ ID NO: 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128 or 130, or is a variant of the SEQ ID NO: 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128 or 130 having an acetate kinase activity; and / or• is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 107, 109, 111 , 113, 115, 117, 119, 121 , 123, 125, 127 or 129, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128 or 130.

21. The recombinant yeast host cell of any one of claims 18 to 20, wherein the heterologous acetyl CoA synthetase is:• of prokaryotic or eukaryotic origin;• encoded by an acs1 gene or an acs2 gene;• derived from Saccharomyces, Zygosaccharomyces, Salmonella, Acetobacter or Escherichia species’,• derived from Saccharomyces cerevisiae, Zygosaccharomyces bailii, Salmonella enterica, Acetobacter aceti or Escherichia coir, and / or• has the amino acid sequence of SEQ ID NO: 132, 134, 136, 138, 140 or 142, or is a variant of the SEQ ID NO: 132, 134, 136, 138, 140 or 142 having an acetyl CoA synthetase activity; and / or• is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 131 , 133, 135, 137, 139 or 141 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 132, 134, 136, 138, 140 or 142.

22. The recombinant yeast host cell of any one of claims 1 to 21 being from the genus Saccharomyces sp.

23. The recombinant yeast host cell of any one of claims 1 to 22 being from the species Saccharomyces cerevisiae.

24. The recombinant yeast host cell of any one of claims 1 to 23 having an increased fermentation kinetic when compared to a control yeast host cell comprising the first and the second engineered metabolic pathways as defined in b) and c) but lacking the upregulated pyruvate kinase as defined in a).

25. The recombinant yeast host cell of any one of claims 1 to 24 producing higher fermentation product yield than a control yeast host cell comprising the first and the second engineered metabolic pathways as defined in b) and c) but lacking the upregulated pyruvate kinase as defined in a).

26. A process for making a fermentation product, the process comprising contacting the recombinant yeast host cell of any one of claims 1 to 25 with a carbohydrate source under a condition allowing the conversion of at least a part of the carbohydrate into a fermentation product.

27. The process of claim 26, wherein the carbohydrate source comprises a corn mash.

28. The process of claims 26 or 27, wherein the recombinant yeast host cell has an increased fermentation kinetic when compared to a control yeast host cell comprising the first and the second engineered metabolic pathway as defined in b) and c) but lacking the upregulated pyruvate kinase as defined in a).

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